TGF-β signaling in health, disease, and therapeutics.

TGF-β signal is transmitted into the cells by TβRI (also known as activin receptor-like kinase 5, ALK5) and TβRII both of which are enzyme-linked receptors with dual specificity of serine/threonine kinase and tyrosine kinase. Studies have revealed that TGF-β1 and TGF-β3 bind TβRII prior to TβRI due to higher affinity, while TGF-β2 binds poorly to both receptors. 12 , 101 , 102 TβRIII, also known as β-glycan, lacks the motifs to directly mediate TGF-β signal transduction. However, TβRIII is able to bind TGF-β especially TGF-β2 with high affinity and thus acts as a co-receptor that presents the ligand to the receptors and further enhances their binding. 101 , 103 – 107 The ligand-receptor interaction subsequently activates the intracellular signaling of TGF-β through a canonical pathway and several non-canonical pathways. The canonical TGF-β signaling is mediated by transcription factors SMADs and thus is also known as the SMAD signaling. Notably, the canonical pathway is under the regulation of various factors that can control the intensity and manner of cellular responses at different levels (Fig. 3 ). Fig. 3 Canonical TGF-β signaling. TGF-β can initially bind to its co-receptor TGF-β receptor III (TβRIII) or directly bind to its receptor TβRII which subsequently recruits TβRI to form a TGF-β-TβRI-TβRII complex. TβRII then actives TβRI through phosphorylation, leading to its dissociation with signaling inhibitor FK506-binding protein 1A (FKBP12) and interaction with signaling effectors receptor-activated SMADs (R-SMADs). R-SMADs which are presented to TβRI by adaptor protein SMAD anchor for receptor activation (SARA) get activated through phosphorylation and undergo oligomerization with common-partner SMAD (co-SMAD). The SMAD oligomers then translocate into the nucleus where they function as transcription factors (TFs), mediating the transcriptional activation or repression of target genes by binding to specific DNA sequences known as SMAD-binding elements (SBEs) and generally in cooperation with other TFs as well as transcriptional cofactors. In this way, TGF-β signaling can activate the expression of inhibitory SMADs (I-SMADs) which in turn function to attenuate the transcriptional regulation mediated by TGF-β signaling through several mechanisms. Moreover, many protein kinases (PKs), protein phosphatases (PPs), and (E3) ubiquitin ligases can also modulate canonical TGF-β signaling through various post-translational modifications of SMADs. (TFBS, TF-binding site) Canonical TGF-β signaling. TGF-β can initially bind to its co-receptor TGF-β receptor III (TβRIII) or directly bind to its receptor TβRII which subsequently recruits TβRI to form a TGF-β-TβRI-TβRII complex. TβRII then actives TβRI through phosphorylation, leading to its dissociation with signaling inhibitor FK506-binding protein 1A (FKBP12) and interaction with signaling effectors receptor-activated SMADs (R-SMADs). R-SMADs which are presented to TβRI by adaptor protein SMAD anchor for receptor activation (SARA) get activated through phosphorylation and undergo oligomerization with common-partner SMAD (co-SMAD). The SMAD oligomers then translocate into the nucleus where they function as transcription factors (TFs), mediating the transcriptional activation or repression of target genes by binding to specific DNA sequences known as SMAD-binding elements (SBEs) and generally in cooperation with other TFs as well as transcriptional cofactors. In this way, TGF-β signaling can activate the expression of inhibitory SMADs (I-SMADs) which in turn function to attenuate the transcriptional regulation mediated by TGF-β signaling through several mechanisms. Moreover, many protein kinases (PKs), protein phosphatases (PPs), and (E3) ubiquitin ligases can also modulate canonical TGF-β signaling through various post-translational modifications of SMADs. (TFBS, TF-binding site) TGF-β ligand initially binds to TβRII monomer to promote its homodimerization or directly binds to pre-existing TβRII homodimer to recruit TβRI for assembly. 108 – 111 This forms a heteromeric TGF-β-TβRI-TβRII complex in which low-affinity TβRI requires high-affinity TβRII to bind TGF-β ligand and constitutively active TβRII requires phosphorylating TβRI to transduce intracellular signal. 112 The phosphorylation of TβRI occurs in its juxtamembrane GS domain at several serine and threonine residues, triggering conformational changes that transform the GS domain from a site that binds the signaling inhibitor known as immunophilin FK506-binding protein 1A (FKBP12) into a binding site for the signaling effectors known as receptor-activated SMADs (R-SMADs). 113 R-SMADs, including SMAD2 and SMAD3, consist of a globular Mad homology 1 (MH1) domain at the N-terminus, a globular MH2 domain at the C-terminus, and a highly flexible long linker region in between. R-SMADs are retained in cytoplasm and presented to TβRI by the adaptor protein known as SMAD anchor for receptor activation (SARA). 114 The R-SMAD MH2 domain then gets phosphorylated at two serine residues in the extreme C-terminal SXS motif by the TβRI kinase domain which is located immediately downstream of the TβRI GS domain. 113 Activated R-SMADs undergo homo-oligomerization or hetero-oligomerization through their MH2 domains upon phosphorylation, and they can also oligomerize with SMAD4, the common-partner SMAD (co-SMAD) which lacks the SXS motif for phosphorylation by TβRI kinase. Notably, studies have suggested that SMAD heterotrimers containing two R-SMADs and one SMAD4 are likely more common and stable than other SMAD oligomers. 115 – 119 Although different SMAD oligomers can vary in function, they all act to regulate the transcription of target genes by binding to DNA after translocating into the nucleus. The MH1 domains of SMAD4, SMAD3, and a specific SMAD2 splicing variant recognize the nucleic acid sequence GTCT or its reverse complement AGAC in double-stranded DNA which are known as the canonical SMAD-binding elements (SBEs). 120 Other SBEs such as the 5GC SBEs including GGCGC and GGCCG have also been discovered, indicating a relatively loose DNA-binding specificity of the SMAD oligomers. 121 However, the binding to a single SBE is so weak that SMAD oligomers generally require interacting with replications of SBE copies as well as other DNA-binding sequence-specific transcription factors to function. 119 , 120 , 122 In fact, many SBE repeats are enriched at the binding sites for SMAD-interacting transcription factors, exactly increasing the binding accessibility, specificity, and affinity of SMAD oligomers associated with specific transcription factors. 123 – 125 Despite a large number of SMAD-interacting transcription factors indicating a huge amount of potential gene targets for canonical TGF-β signaling, the dominant effects are generally determined by the master transcription factors in specific cell types and contexts which contribute to the complexity and variability of cellular responses to TGF-β. 125 TGF-β and many other factors can induce the expression of SMAD6 and SMAD7 which function to inhibit TGF-β signaling and thus are known as I-SMADs. 126 , 127 Unlike R-SMADs, I-SMADs lack the N-terminal MH1 domain and the C-terminal SXS motif, however, they retain the C-terminal MH2 domain which can competitively bind to activated receptor TβRI to inhibit the phosphorylation of R-SMADs. 128 , 129 Through some extra mechanisms, SMAD7 confers greater abilities in suppressing TGF-β signaling than SMAD6 does. 130 For example, SMAD7 recruits E3 ubiquitin ligases such as SMAD ubiquitination regulatory factors (SMURFs) and neural precursor cell expressed, developmentally downregulated 4-like (NEDD4L) to TβRI, R-SMADs, and co-SMAD to mediate the proteasomal and lysosomal degradation of these TGF-β signaling components. 131 – 135 SMAD7 can also trigger the dephosphorylation of TβRI by recruiting protein phosphatase 1 (PP1) to the receptor. 136 Moreover, with its MH2 domain, SMAD7 can oligomerize with R-SMADs to compete with co-SMAD 133 and can bind to specific DNA sequences to disrupt the formation of the transcriptional SMAD-DNA complex. 137 Taken together, TGF-β signaling induces I-SMADs to form a negative feedback loop of itself. Transcriptional cofactors are actively recruited to the transcriptional SMAD complex to regulate its activity. Notably, many of these transcriptional cofactors have histone modification activity and thus enable TGF-β signaling to trigger epigenetic changes. Histone acetyltransferases (HATs) such as p300, cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB)-binding protein (CBP), p300/CBP-associated factor (PCAF), and general control non‐repressed protein 5 (GCN5) act as the transcriptional coactivators of SMADs by increasing the accessibility to DNA. 138 – 141 The interaction between p300/CBP and doubly phosphorylated R-SMADs requires SMAD4 for stabilization and is critical for SMAD-mediated transcriptional activation. Other SMAD coactivators include melanocyte-specific gene 1 (MSG1), 142 zinc finger E-box-binding homeobox 1 (ZEB1), 143 , 144 and the histone methyltransferase (HMT) known as SET domain-containing protein 7 (SETD7). 145 Contrary to HATs, histone deacetylases (HDACs) generally act as the transcriptional corepressors of SMADs by decreasing the accessibility to DNA. SMAD3 can directly recruit HDAC4 and HDAC5 to gene promoters to inhibit the function of transcription factors via histone deacetylation. 146 SMADs can also associate with HDACs through interaction with other corepressors such as TGF-β-induced factor (TGIF), 147 ecotropic viral integration site 1 (EVI1), 148 , 149 Sloan-Kettering Institute proto-oncogene (SKI), 150 – 152 as well as SKI-related novel gene N (SNO). 153 Other transcriptional corepressors of SMADs include cellular-myelocytomatosis viral oncogene (MYC), 154 SMAD nuclear-interacting protein 1 (SNIP1), 155 ZEB2, 143 , 156 and HMTs such as suppressor of variegation 3-9 homolog 1 (SUV39H1) and SET domain bifurcated 1 (SETDB1) which can both trigger the methylation of histone 3 lysine 9 (H3K9) at gene promoters. 157 , 158 Post-translational modifications can also regulate the functions of SMADs. Apart from TβRI kinase which phosphorylates R-SMADs in their C-terminal SXS motif to mediate their activation, many other protein kinases such as mitogen-activated protein kinase kinase kinase 1 (MAPKKK1), 159 p38 MAPK, 160 c-Jun N-terminal kinase (JNK), 161 extracellular signal-regulated kinase (ERK), 162 – 164 rat sarcoma (RAS) homolog (Rho)-associated coiled-coil-containing protein kinase (ROCK), 160 glycogen synthase kinase (GSK)-3β, 165 – 167 calcium/calmodulin-dependent protein kinase II (CAMK2), 168 protein kinase C (PKC), 169 PKG, 170 and several cyclin-dependent kinases (CDKs) 167 , 171 , 172 can phosphorylate R-SMADs as well as co-SMAD at many different sites to enhance or attenuate SMAD activity. Meanwhile, the various phosphorylation of SMADs can be reversed by phosphatases. Several nuclear phosphatases known as the small C-terminal domain phosphatases (SCPs) can specifically dephosphorylate the linker region and MH1 domain of R-SMADs, 173 , 174 whereas protein phosphatase, magnesium/manganese-dependent 1A (PPM1A), 175 myotubularin-related protein 4 (MTMR4), 176 and protein phosphatase 2A (PP2A) 177 catalyze the dephosphorylation of the C-terminal SXS motif to terminate the signaling and promote the dissociation and cytoplasmic localization of SMADs. Furthermore, SMADs can be ubiquitinated and deubiquitinated respectively by E3 ubiquitin ligases and deubiquitylating enzymes (DUBs). The E3 ubiquitin ligases that can mediate SMAD ubiquitination include SMURFs, 135 , 178 – 180 NEDD4L, 134 , 181 WW domain-containing proteins (WWPs), 182 – 184 really interesting new gene (RING) finger protein 111 (RNF111), 185 C-terminus of heat shock protein (HSP) 70-interacting protein (CHIP), 186 itchy (ITCH) E3 ubiquitin ligase, 187 and S-phase kinase-associated protein (SKP)-cullin-F-box (SCF) E3 ubiquitin ligase complex. 188 , 189 The ubiquitination generally leads to the proteasomal degradation of SMADs, but in some cases, it also exerts non-degradative effects on SMAD activity. 190 Notably, the degradative ubiquitination of R-SMADs by NEDD4L requires the phosphorylation of the R-SMAD linker by CDK8/9 and GSK-3 in sequence to create binding sites for the E3 ubiquitin ligase. 171 , 181 , 191 Apart from the SMAD-dependent pathway, TGF-β can also signal through SMAD-independent pathways to activate ERK signaling, Rho guanosine triphosphatase (GTPase) signaling, p38 MAPK signaling, JNK signaling, nuclear factor-κB (NF-κB) signaling, phosphatidylinositol 3-kinase (PI3K)/AKR mouse thymoma proto-oncogene (AKT) signaling, as well as Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. These non-canonical TGF-β signaling pathways are involved in an extensive range of cellular events, greatly expanding the participation of TGF-β signaling in health and disease (Fig. 4 ). Fig. 4 Non-canonical TGF-β signaling. TGF-β can signal through non-canonical pathways to activate extracellular signal-regulated kinase (ERK) signaling, rat sarcoma (RAS) homolog (Rho)-guanosine triphosphatase (GTPase) signaling, p38 mitogen-activated protein kinase (MAPK) signaling, c-Jun N-terminal kinase (JNK) signaling, nuclear factor-κB (NF-κB) signaling, phosphatidylinositol 3-kinase (PI3K)/AKR mouse thymoma proto-oncogene (AKT) signaling, as well as Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. These non-canonical TGF-β signaling pathways are actively involved in an extensive range of cellular events. (RAF, RAS-associated factor; MEK, MAPK/ERK kinase; ROCK1, Rho-associated coiled-coil-containing protein kinase 1; LIMK2, LIM domain kinase 2; TRAF, tumor necrosis factor (TNF) receptor-associated factor; TAK1, TGF-β-activated kinase 1; MKK, MAPK kinase; IKK, NF-κB inhibitor (IκB) kinase; GSK-3β, glycogen synthase kinase-3β; MTOR, mechanistic target of rapamycin; FOXO, forkhead box O; S6K, S6 kinase; 4EBP1, 4E-binding protein 1) Non-canonical TGF-β signaling. TGF-β can signal through non-canonical pathways to activate extracellular signal-regulated kinase (ERK) signaling, rat sarcoma (RAS) homolog (Rho)-guanosine triphosphatase (GTPase) signaling, p38 mitogen-activated protein kinase (MAPK) signaling, c-Jun N-terminal kinase (JNK) signaling, nuclear factor-κB (NF-κB) signaling, phosphatidylinositol 3-kinase (PI3K)/AKR mouse thymoma proto-oncogene (AKT) signaling, as well as Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. These non-canonical TGF-β signaling pathways are actively involved in an extensive range of cellular events. (RAF, RAS-associated factor; MEK, MAPK/ERK kinase; ROCK1, Rho-associated coiled-coil-containing protein kinase 1; LIMK2, LIM domain kinase 2; TRAF, tumor necrosis factor (TNF) receptor-associated factor; TAK1, TGF-β-activated kinase 1; MKK, MAPK kinase; IKK, NF-κB inhibitor (IκB) kinase; GSK-3β, glycogen synthase kinase-3β; MTOR, mechanistic target of rapamycin; FOXO, forkhead box O; S6K, S6 kinase; 4EBP1, 4E-binding protein 1) As a dual-specificity kinase, TβRI can phosphorylate at its tyrosine residues to activate ERK signaling upon TGF-β stimulation. 192 In this case, TβRI with tyrosine kinase activity initially phosphorylates the adapter protein known as sarcoma (SRC) homology and collagen A (SHCA) which subsequently forms a complex with growth factor receptor-bound protein 2 (GRB2) and son of sevenless homolog (SOS). The SHCA-GRB2-SOS complex then initiates a canonical MAPK signaling cascade which involves the sequential activation of RAS, the MAPKKK known as RAS-associated factor (RAF), the MAPKK known as MAPK/ERK kinase (MEK), and eventually, the ERK MAPK. Activated ERK is known to regulate various biological events including cell survival, proliferation, differentiation, adhesion, migration, as well as metabolism, and is implicated in a spectrum of diseases such as developmental disorders, chronic inflammation, neurodegeneration, obesity, and cancers. 193 , 194 Rho GTPases such as RHO, RAS-related C3 botulinum toxin substrate 1 (RAC1), and cell division cycle 42 (CDC42) play a central role in the organization and dynamics of the actin cytoskeleton. They are activated by guanine nucleotide exchange factors (GEFs) through the exchange of a bound GDP for GTP. 195 TGF-β can trigger RHO activation in a rapid SMAD-independent manner or by inducing a GEF known as neuroepithelial cell transforming 1 (NET1) through SMAD and MEK/ERK pathways. 196 – 200 RHO then activates its key effector ROCK1 which further mediates the phosphorylation of LIM domain kinase 2 (LIMK2). Activated LIMK2 subsequently phosphorylates cofilin to inhibit its function as a constitutive actin-depolymerizing factor, leading to the reorganization of the actin cytoskeleton in the end. 201 – 203 Additionally, TGF-β-triggered RHO/ROCK1 signaling can contribute to ERK phosphorylation, 204 , 205 and besides RHO, TGF-β can also activate the signaling of other Rho GTPases such as RAC1 202 and CDC42. 206 Besides the regulation of cell morphogenesis, adhesion, and movement, Rho GTPase signaling is also known to participate in transcriptional regulation, cell cycle progression, vesicular trafficking, and pathological processes such as fibrosis, inflammation, wound repair, and tumor development. 207 , 208 TGF-β can activate the signaling of another two MAPKs known as p38 and JNK through a receptor kinase-independent mechanism which is different from that of ERK signaling. TGF-β-activated TβR complex can recruit tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) and TRAF6 to trigger their lysine 63 (K63)-linked polyubiquitination. With E3 ubiquitin ligase activity, polyubiquitinated TRAF then attaches the polyubiquitin chain on the MAPKKK known as TGF-β-activated kinase 1 (TAK1) which subsequently gets activated and phosphorylates several MAPKKs (MKKs). 209 – 211 As a result, MKK3 and MKK6 specifically trigger the activation of p38 while MKK4 mediates the phosphorylation of both p38 and JNK. TGF-β-activated Rho GTPases such as RHOA, RAC1, and CDC42 can also contribute to p38 and JNK activation. 204 , 212 – 216 Both the two MAPKs regulate a series of biological events to respond to all kinds of environmental and intracellular stresses, meanwhile, they engage actively in embryonic development, metabolic regulation, neuronal functions, immunological actions, as well as tumor development. 217 – 220 Additionally, TGF-β-activated TRAF/TAK1 signaling, RHO/ROCK1 signaling, and PI3K/AKT signaling can also lead to the phosphorylation of NF-κB inhibitor (IκB) kinase (IKK). 221 – 224 Activated IKK then triggers the phosphorylation of IκB which subsequently gets polyubiquitinated and degraded while releasing active NF-κB for nuclear translocation. 221 NF-κB as a transcription factor can regulate hundreds of genes involved in cell survival, proliferation, metabolism, and immunity in particular. 225 – 227 The TβR complex can activate the lipid kinase PI3K upon TGF-β stimulation, either via the kinase activity of TβRI or through the recruitment of TRAF6, which polyubiquitylates PI3K regulatory subunit p85α independent of the receptor kinase. 228 , 229 Activated PI3K then phosphorylates phosphoinositide phosphatidylinositol-4,5-bisphosphate (PIP2) into phosphatidylinositol-3,4,5-trisphosphate (PIP3) which further triggers the phosphorylation of AKT. 228 , 230 Activated AKT targets plenty of substrates, including mechanistic target of rapamycin (MTOR), 231 , 232 GSK-3β, 233 and several forkhead box O (FOXO) transcription factors. 234 Among them, MTOR is the most common downstream effector of AKT, and ribosomal protein S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein 1 (4EBP1) are the best-characterized downstream effectors of MTOR. In general, the consequences of PI3K/AKT signaling include diverse cellular responses such as survival, metabolism, growth, proliferation, and differentiation. 235 TGF-β is found to induce JAK1 and JAK2 activation respectively in hepatic stellate cells (HSCs) and fibroblasts. In these cases, activated JAK triggers the phosphorylation of STAT3 which functions to mediate the fibrogenic effects of TGF-β, including increased cell proliferation, myofibroblast (MF) differentiation, ECM production, α-smooth muscle actin (α-SMA) expression, and stress fiber formation. 236 – 238 Like other signaling pathways, JAK/STAT signaling can also drive many physiological and pathological events, including development, metabolism, immunity, wounding, and cancers. 239

Dysfunctional TGF-β signaling can play key roles in numerous pathological processes, contributing to the disorders of developmental defects, aberrant healing, fibrotic diseases, inflammatory diseases, infectious diseases, as well as tumors (Fig. 6 ). Fig. 6 TGF-β signaling in disease. Dysfunctional TGF-β signaling is involved in numerous pathological processes. a Mutations that lead to decreased or increased TGF-β signaling can cause various developmental defects. b Deficient TGF-β signaling contributes to wound chronicity while excess TGF-β signaling leads to wound scarring and tissue fibrosis by stimulating ECM deposition through fibroblast activation and EMT/EndMT. c Dysfunctional TGF-β signaling exacerbates tissue injuries in inflammatory diseases and infectious diseases by promoting inflammation, pathogen infection, and tissue remodeling. d Aberrant TGF-β signaling is implicated in all aspects of tumor development including tumorigenesis, tumor growth, tumor invasion, tumor metastasis, as well as tumor microenvironment (TME) remodeling. (CTGF, connective tissue growth factor; IFN-γ, interferon-γ; IL-6, interleukin-6; solid arrows from TGF-β indicate excessive TGF-β signaling, dashed arrows from TGF-β indicate deficient TGF-β signaling) TGF-β signaling in disease. Dysfunctional TGF-β signaling is involved in numerous pathological processes. a Mutations that lead to decreased or increased TGF-β signaling can cause various developmental defects. b Deficient TGF-β signaling contributes to wound chronicity while excess TGF-β signaling leads to wound scarring and tissue fibrosis by stimulating ECM deposition through fibroblast activation and EMT/EndMT. c Dysfunctional TGF-β signaling exacerbates tissue injuries in inflammatory diseases and infectious diseases by promoting inflammation, pathogen infection, and tissue remodeling. d Aberrant TGF-β signaling is implicated in all aspects of tumor development including tumorigenesis, tumor growth, tumor invasion, tumor metastasis, as well as tumor microenvironment (TME) remodeling. (CTGF, connective tissue growth factor; IFN-γ, interferon-γ; IL-6, interleukin-6; solid arrows from TGF-β indicate excessive TGF-β signaling, dashed arrows from TGF-β indicate deficient TGF-β signaling) Loss of TβRI or TβRII functions due to homozygous mutations generally results in embryonic lethality in mice due to defects in the hematopoiesis and vasculogenesis of yolk sac. 451 , 452 However, the lack of different TGF-β isoforms can lead to distinct phenotypes in mice, consistent with the isoform-specific roles of TGF-β in embryonic development. TGF-β1-knockout mice show no gross developmental abnormalities in spite of the defective hematopoiesis and vasculogenesis in yolk sac during embryonic development. 452 – 454 In contrast, TGF-β2-knockout mice exhibit perinatal mortality and a wide range of developmental defects in heart, lungs, bones, eyes, inner ears, craniofacial structures, urogenital organs, and hair follicles. 290 , 455 – 458 TGF-β3-knockout mice also die shortly after birth but show no detectable abnormalities except for cleft palate and abnormal lung development. 459 , 460 Notably, palatal shelves that fail to elevate in TGF-β2-knockout mice undergo elevation in TGF-β3-knockout mice but still fail in fusion. 455 , 459 , 460 Also, branching morphogenesis and respiratory epithelial cell differentiation which appear normal in the lungs of TGF-β2-knockout mice are defective in TGF-β3-knockout mice. 455 , 459 In humans, loss-of-function mutations of a single TGF-β signaling component such as TGF-β2, 461 – 463 TGF-β3, 464 – 466 TβRI, 467 – 469 TβRII, 470 – 472 SMAD2, 473 – 475 or SMAD3 476 – 478 can cause Loeys-Dietz syndrome (LDS), an autosomal dominant connective tissue disorder with a range of cardiovascular, skeletal, craniofacial, and cutaneous manifestations. LDS patients typically present with features including congenital heart defects, aneurysms, arterial tortuosity and dissections, skeletal overgrowth, cervical spine instability, clubfoot deformity, craniosynostosis, hypertelorism, bifid uvula, cleft palate, thin skin, and mental retardation. Dermal fibroblasts derived from LDS patients demonstrate impaired deposition of extracellular collagen and elastin, suggesting a possible mechanism of the connective tissue defects of the patients. 479 , 480 However, the aortic tissues of LDS patients show increased accumulation of collagen, elevated expression of connective tissue growth factor (CTGF), and enhanced activity of non-mutant TGF-β signaling components. 461 – 463 , 465 , 467 , 468 , 475 , 476 , 481 , 482 Therefore, primary downregulation and compensatory upregulation of TGF-β signaling are both responsible for the abnormalities of LDS. Excessive TGF-β signaling can also act as a primary pathogenic factor in developmental defects. In mice, overexpression of TGF-β or SMAD can lead to developmental abnormalities in several tissues, such as skin, 483 , 484 bones, 485 eyes, 486 lungs, 487 , 488 mammary glands, 489 – 491 salivary glands, 492 and central nervous system. 493 In humans, Camurati-Engelmann disease (CED), a progressive bone dysplasia inherited in an autosomal dominant manner, is ascribed to mutations of TGF-β1, which lead to increased TGF-β1 activation and signaling. 494 , 495 This disease is characterized by hyperostosis and sclerosis of the long bones and the skull. 496 , 497 Studies on CED have suggested that hyperactive TGF-β1 in the bone microenvironment can induce osteoclasts and osteoblasts to increase but cluster in separated areas, uncoupling bone resorption and formation to cause bone remodeling defects. 494 , 498 , 499 Dysregulated TGF-β signaling can contribute to the tissue damage in aberrant healing and fibrotic diseases which are caused by all kinds of injuries such as wounding, burns, radiation, infection, and inflammation. The lack of TGF-β and TβR expression is commonly found in the chronic wounds in patients, indicating that deficient TGF-β signaling may lead to wound chronicity and even unhealing. 500 – 505 However, in vivo studies in mice have reported quite complicated findings. An activating mutation of TβRI can lead to a regenerative healing phenotype which enables rapid regeneration of normal tissues with differentiated structures instead of scar formation in ear punch wounds. 506 Paradoxically, overexpression of TGF-β1 in keratinocytes accelerates the re-epithelialization in partial-thickness cutaneous wounds but slows that of full-thickness cutaneous wounds. 507 , 508 In TGF-β1-deficient mice, the healing of full-thickness cutaneous wounds is initially normal but ultimately damaged by severe inflammatory diseases. 509 In immunodeficient mice without inflammatory diseases, the lack of TGF-β1 still leads to significant delays in each healing stage of full-thickness cutaneous wounds. 510 However, loss of TGF-β signaling in keratinocytes due to expression of dominant negative TβRII leads to increased proliferation and reduced apoptosis, thus facilitating the re-epithelialization in full-thickness cutaneous wounds. 511 Furthermore, cutaneous wound healing is accelerated in mice lacking SMAD3 but is aberrant in mice lacking SMAD4 exclusively in keratinocytes. 512 , 513 In contrast to chronic wounds, hypertrophic scars and keloids both characterized by overabundant ECM deposition are the results of hyperactive cutaneous wound healing. In fact, the expression of TGF-β and TβR which decreases eventually in normal cutaneous wounds remains elevated in hypertrophic scars and keloids. 514 – 518 In contrast to normal cutaneous fibroblasts, both keloid fibroblasts and hypertrophic scar fibroblasts are significantly higher in collagen production, however, only keloid fibroblasts exhibit increased sensitivity to TGF-β stimulation. 519 For keloid fibroblasts, overexpressed TGF-β can promote the resistance to apoptosis, the ability of proliferation, the conversion to MFs, and the expression of CTGF and VEGF, thus contributing to the ECM deposition, focal adhesion, fibrous growth, and angiogenesis in keloid tissues. 518 , 520 – 523 Besides wounding, other forms of injurious stimulation can also cause excessive ECM deposition in different kinds of tissues, leading to fibrotic diseases, which are closely associated with the hyperactivity of TGF-β signaling. TGF-β expression is significantly elevated in fibrotic lungs in various cases such as idiopathic pulmonary fibrosis (IPF) and cystic fibrosis (CF). 524 – 528 In situ hybridization and immunohistochemical staining suggest that alveolar macrophages and epithelial cells are likely the major sources of TGF-β which contribute to the fibrosis of lungs. 526 – 528 In vitro studies show that TGF-β1 can trigger the EMT of alveolar epithelial cells and enhance the activity of lung fibroblasts to mediate fibrogenic effects. 529 – 532 Transgenic expression of TGF-β1 in murine and rat lungs induces pulmonary fibrosis which is accompanied by alveolar EMT, MF differentiation, and mononuclear-rich inflammation. 532 – 535 Interestingly, the suppression of TGF-β1, the deletion of TβRII, the ablation of SMAD3, the upregulation of SMAD7, but the administration of TGF-β3 can all significantly protect mice from experimentally induced pulmonary fibrosis. 535 – 539 Similarly, the fibrotic kidneys of human glomerulonephritis, IgA nephropathy, diabetic nephropathy, lupus nephritis, as well as renal allografts in chronic rejection all show significant increases in three TGF-β isoforms in the glomeruli and tubulointerstitium where ECM deposition and PAI-1 production is closely related to the expression of TGF-β1 isoform in particular. 540 – 543 In vitro, TGF-β1 stimulates kidney fibroblasts, mesangial cells, glomerular epithelial cells, and tubular epithelial cells to produce several ECM components and remodelers such as collagen, fibronectin, laminin, proteoglycan, MMP, and TIMP. 544 – 550 TGF-β1 also contributes to the EMT induction and MF differentiation in renal fibrosis. 550 Transgenic mice that have increased levels of TGF-β1 in plasma develop progressive renal disease characterized by glomerulosclerosis and tubulointerstitial fibrosis with TIMP overexpression and ECM deposition in sub-endothelial and mesangial locations. 551 , 552 In fibrotic livers, TGF-β1 expression increases markedly with fibrogenic activity. 553 – 556 Induction of TGF-β1 expression in murine livers leads to hepatic fibrosis characterized by prominent ECM deposition in peri-sinusoidal areas with activation of HSCs and apoptosis of hepatocytes. 557 , 558 Notably, activated HSCs which play a major role in hepatic fibrosis can provide an important source of TGF-β, 559 while overproduced TGF-β can in turn activate several signaling pathways such as those of SMAD, MEK, JNK, PI3K, and JAK/STAT in HSCs to contribute to their functions. 236 , 237 As for the cardiovascular system, TGF-β is also elevated during myocardial fibrosis, valve fibrosis, and arteriosclerosis, generally attributed to the expression by SMCs, fibroblasts, endothelial cells, and inflammatory cells such as macrophages. 560 – 569 On one hand, TGF-β can stimulate cardiovascular fibroblasts to differentiate into MFs and produce ECM components and remodelers, 562 , 563 , 570 – 573 on the other hand, it can also stimulate endothelial cells to undergo EndMT to induce their fibrogenic phenotype. 569 , 574 , 575 Furthermore, TGF-β is widely involved in the fibrosis of many other tissues and diseases as in the cases of cutaneous fibrosis, 576 , 577 muscular fibrosis, 578 pancreatic fibrosis, 579 – 582 myelofibrosis, 583 , 584 adenomyosis, 585 autoimmune diseases, 238 , 527 , 573 , 586 – 589 and infectious diseases. 590 – 593 Inflammatory diseases and infectious diseases can demonstrate aberrant immune responses and various tissue injuries which usually implicate the dysfunction of TGF-β signaling. Since TGF-β acts as a negative regulator to maintain immune homeostasis, deficient TGF-β signaling can lead to hyperactive immune responses, contributing to the pathology of numerous inflammatory diseases. TGF-β1-null mice initially appear normal after birth but soon develop a rapid wasting syndrome accompanied by a multifocal inflammatory disease which leads to organ failure and early death by 3-4 weeks of age. 453 , 594 – 596 Many organs in these mice, including heart, lungs, stomach, liver, pancreas, and muscles, all exhibit massive infiltration of inflammatory cells such as lymphocytes, macrophages, and granulocytes. Moreover, their total numbers of blood leukocytes increase mainly due to the elevated absolute numbers of neutrophils and monocytes, while their levels of autoantibodies, MHC molecules, and inflammatory cytokines such as IFN-γ, TNF-α, and CCL3 also rise correspondingly in serum or tissues. In the absence of any pathogens, the inflammatory diseases in TGF-β1-knockout mice actually resemble a special group of inflammatory diseases known as autoimmune diseases, which are characterized by dysregulated immune responses attacking self-tissues. In fact, even cell type-specific loss of TGF-β signaling can lead to the development of various autoimmune diseases in mice. 597 – 604 In patients with autoimmune diseases such as systemic lupus erythematosus (SLE), 605 – 607 systemic sclerosis (SSc), 608 – 611 rheumatoid arthritis (RA), 612 – 614 Sjögren’s syndrome, 586 , 614 – 616 Crohn’s disease, 587 , 617 – 619 ulcerative colitis (UC), 617 – 622 autoimmune hepatitis (AIH), 623 , 624 and Hashimoto’s thyroiditis (HT), 606 , 625 , 626 the levels of TGF-β or TβR in tissues or circulation are associated with the presence, activity, and severity of the diseases. Notably, although all these diseases show correlations with dysregulated TGF-β signaling, their correlations with TGF-β levels can be either positive or negative. Some cases of the diseases are likely caused by insufficient TGF-β expression and thus exhibit decreased TGF-β production. 619 , 622 , 627 – 630 In other cases, however, the autoimmune inflammation is likely attributed to impaired cell responsiveness to TGF-β especially due to deficient TβR functions, therefore, TGF-β production is elevated as a compensatory response. 624 , 628 , 631 – 635 Allergic diseases, including asthma, allergic rhinitis, food allergy, and atopic dermatitis, are another group of inflammatory diseases that are caused by aberrant immune responses to harmless environmental antigens. TGF-β production is increased in the airways and serum of asthmatic patients and is further increased after allergen exposure, disease progression, or certain treatments. 636 – 646 Bronchial epithelial cells, fibroblasts, SMCs, eosinophils, neutrophils, and macrophages can all contribute to the excessive TGF-β production in asthmatic patients. 641 – 650 However, the functions of TGF-β are seemingly contradictory in the context of allergic airway inflammation, for TGF-β can either enhance or suppress the activity of eosinophils, lymphocytes, macrophages, and mast cells in asthma. 648 , 651 – 661 Nevertheless, it is clear that TGF-β can promote asthmatic airway remodeling by inducing airway EMT, 662 , 663 ECM production, 649 , 650 MF differentiation, 664 , 665 and smooth muscle hyperplasia. 647 In patients with allergic rhinitis, TGF-β levels in serum are found dependent on allergen exposure, while TGF-β and TβR expression in nasal mucosa is noticed correlated with intra-epithelial mast cell abundance. 666 – 668 In fact, allergen challenge can activate TGF-β signaling in the mast cells and epithelial cells in nasal mucosa which may contribute to the mast cell accumulation and goblet cell hyperplasia in allergic rhinitis. 669 , 670 Allergen challenge can also induce the loss of TGF-β1-expressing Bregs and Tregs which function to suppress the inflammatory Th2 responses of allergic rhinitis. However, with prolonged challenging time, the proportion of TGF-β1-expressing Bregs and Tregs can gradually recover to reconstitute the immune homeostasis in nasal mucosa. 671 Similarly, TGF-β can inhibit the Th2 responses of food allergy by promoting Treg activity in the intestines. 603 , 672 , 673 Therefore, reduced TGF-β1 expression in the intestinal epithelial cells and mononuclear cells of patients with food allergy can partially account for the development of the disease. 603 , 674 Moreover, TGF-β can inhibit the pathology of atopic dermatitis by suppressing B cell maturation, mast cell activation, eosinophil infiltration, as well as the secretion of IgE, TNF-α, and histamine by those cells. 675 – 677 Aberrant TGF-β expression or attenuated cell responsiveness discovered in patients with atopic dermatitis may play a key role in the disorder. 678 – 680 Furthermore, TGF-β signaling is implicated in the pathology of other inflammatory diseases and inflammation-related diseases such as bronchitis, 642 pancreatitis, 681 – 683 glomerulonephritis, 684 , 685 osteomyelitis, 686 arthritis, 687 diabetes, 688 and Alzheimer’s disease (AD). 689 , 690 Infectious diseases caused by different kinds of pathogenic organisms can result in tissue damage due to diverse pathogen virulence and dysregulated host responses. TGF-β can function to reduce pathogen burdens as well as tissue injuries in some cases of infection. In patients with H1N1 influenza A virus sepsis, blood TGF-β levels are negatively correlated with clinical severity scores on admission. 691 Consistently, increased TGF-β activity in mice confers resistance against lethal influenza infection due to reductions in both viral titers and pulmonary inflammation. 692 , 693 TGF-β expression also prevents mice from coxsackievirus-induced myocarditis and type 1 diabetes in a Treg-dependent manner. 694 , 695 Moreover, TGF-β acts as a pro-survival factor to protect murine neurons and intestinal epithelial cells against cell death during reovirus infection. 696 , 697 As for bacterial infection, TGF-β can attenuate sepsis-induced tissue injuries through mechanisms involving the induction of Tregs. 698 It also enhances the pathogen clearance and host resistance of mice during the infection of Streptococcus pneumoniae, 699 Streptococcus pyogenes, 700 Listeria monocytogenes, 701 and Yersinia enterocolitica, 702 likely, by suppressing IFN-γ, TNF-α, and IL-6 production while promoting Th17 and Treg responses. In rats with pulmonary cryptococcosis, TGF-β reduces fungal burdens by promoting the lysozyme secretion of macrophages, meanwhile, it also limits inflammation by inhibiting macrophage phagocytosis, chemokine production, and oxidative burst. 703 Moreover, TGF-β can be protective during parasitic infection. The lack of TGF-β exacerbates the severity of murine malaria infection, whereas TGF-β treatment, in contrast, suppresses plasmodium proliferation and prolongs mice survival with decreased TNF-α and increased IL-10 in serum. 704 During Trypanosoma congolense infection, exogenous TGF-β1 confers early protection against parasitemia, anemia, splenomegaly, and mortality due to enhanced macrophage activity and Th1 responses which are characterized by increased NO, IFN-γ, TNF-α, IL-12, and IgG2a production. 705 During Toxoplasma infection, TGF-β can prevent tissue damage by reducing inflammatory cell infiltration and cytokine production, while it can also improve the outcomes of infection-related abnormal pregnancy by promoting Treg functions and suppressing NK cytotoxicity. 706 – 709 Furthermore, TGF-β can prevent the lung injuries during hookworm infection by inducing the immunosuppressive activity of myeloid cells to reduce Th2 responses. 710 In other cases of infection, however, TGF-β can turn to facilitate pathogen infection and tissue injuries. In clinical patients, circulating TGF-β1 levels are positively correlated with the severity and mortality of severe community-acquired pneumonia (CAP) 711 and sepsis-induced acute respiratory distress syndrome (ARDS). 712 Increased TGF-β production can impair the anti-bacterial functions of neutrophils, uncouple the cytokine production and glycolysis of macrophages, and suppress the IL-2 expression and proliferation of T cells to participate in the pathology of sepsis. 713 – 715 As for bacterial infection in local tissues, on one hand, TGF-β can upregulate fibronectin and integrins in hosts to promote bacterial adhesion and invasion, 716 , 717 on the other hand, it can attenuate anti-infectious innate responses and Th1 responses while inducing immunotolerant Treg responses to facilitate the immune escape of the pathogens. 718 – 722 TGF-β-mediated immunosuppression can also contribute to viral infection, as elevated TGF-β expression during viral infection not only impairs early innate immunity such as IFN responses, NK functions, and macrophage activity but also suppresses the adaptive immune responses of T cells and B cells. 428 , 723 – 731 Notably, TGF-β can also enhance viral infection through certain pathogen-specific mechanisms as in the cases of human immunodeficiency virus type 1 (HIV-1) infection, 732 – 734 human T-cell leukemia virus type I (HTLV-I) infection, 735 hepatitis C virus (HCV) infection, 736 Zika virus (ZIKV) infection, 737 as well as rubella virus (RuV) infection. 738 Furthermore, TGF-β can promote the survival and growth of parasites in hosts through downregulation of NO, IFN-γ, TNF-α, IL-6, IL-17, and Th17 cells as well as upregulation of IL-4, IL-10, and Treg cells, contributing to the infection of Fasciola hepatica, 739 Echinococcus multilocularis, 740 Toxoplasma gondii, 741 Leishmania, 742 and Plasmodium. 743 , 744 It is generally accepted that TGF-β acts as a tumor suppressor during the early stages of tumorigenesis but turns into a tumor promotor at later stages of tumor development. Evidence from animal models firmly establishes the suppressor role of TGF-β signaling in early tumorigenesis. TGF-β and its receptors can be strongly induced in the murine epidermis upon exposure to carcinogens that tend to disrupt tissue homeostasis and cause oncogenic transformation. 745 , 746 Increased TGF-β expression in murine epidermis can potently attenuate cell proliferation and confer resistance to hyperproliferation induced by carcinogens. 316 , 746 , 747 Similarly, in murine mammary epithelia, the overexpression of TGF-β or TβR can result in remarkable protection from carcinogen-induced tumorigenesis with reduced premalignant lesions, prolonged tumor latency, and decreased cancer incidence. 25 , 491 , 748 – 750 Such tumor-inhibitory effects by TGF-β signaling are attributed to the early apoptosis of differentiating cells and, more importantly, the premature senescence of stem cells which reduces the reproductive capacity of the mammary epithelia and thus decreases the frequency with which transforming mutations may occur and be fixed in the cell population. 491 , 748 In contrast, loss of TGF-β signaling can be an early event that contributes to tumorigenesis. In clinical patients, heterogeneous patterns of TβRII expression in normal breast lobular units as well as loss of TβRII expression in breast epithelial hyperplastic lesions are both associated with increased risks of invasive breast cancer. 751 More convincing evidence is provided by germline mutations of TGF-β signaling components which show strong correlations with increased risks of tumorigenesis. Loss-of-function TβRI mutations can result in an autosomal dominant skin cancer condition known as multiple self-healing squamous epithelioma (MSSE) or Ferguson-Smith disease (FSD) which is characterized by multiple squamous-carcinoma-like skin tumors that invade locally and then regress spontaneously after several months. 752 , 753 Inactivating TβRII mutations are considered causative of some cases of hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome, an autosomal dominant cancer predisposition syndrome, by impairing cell growth inhibition in response to TGF-β. 754 Moreover, germline mutations of SMAD4 are responsible for juvenile polyposis, an autosomal dominant syndrome predisposing to gastrointestinal hamartomatous polyps and cancers. 755 , 756 Mechanically, impaired TGF-β signaling can cause serious disturbance to tissue homeostasis, thus largely facilitating the development of pre-neoplastic lesions, as well as subsequent tumors, as shown in different murine tissues with deficiencies in the activity of TGF-β, 757 , 758 TβR, 749 , 759 – 770 or SMAD. 657 , 771 – 776 Among them, TβR-deleted murine epithelia exhibit significant reductions in p15 and p21 and remarkable increases in MYC expression and RAS/ERK signaling, accompanied by elevated cell proliferation, reduced cell apoptosis, and enhanced cell malignant transformation to become tumorigenic. 762 – 765 Furthermore, TGF-β can provide additional protection against tumorigenesis by controlling pathogen infection, 777 inhibiting excessive inflammation, 778 – 781 reducing genomic instability, 782 inducing replicative senescence, 783 and regulating epithelial-mesenchymal interaction. 784 TGF-β can inhibit tumor growth by triggering cytostasis and apoptosis through similar mechanisms as it does in cells from normal tissues. In tumor cells, TGF-β signaling induces cell cycle arrest by targeting effectors, such as p15, 354 , 356 p21, 355 , 785 , 786 p27, 361 MYC, 363 ID, 787 and CDC25A, 374 , 786 while it also induces apoptotic cell death through effectors including CTGF, 788 programmed cell death 4 (PDCD4), 789 Fas receptor, 790 death-associated protein kinase (DAPK), 791 DAXX, 383 IκB-α, 384 , 386 sex-determining region Y (SRY)-box 4 (SOX4), 792 ARTS, 382 TIEGs, 793 as well as several BCL-2 family members. 794 – 800 Consistently, primary tumors induced from murine tissues with intact TGF-β signaling pathways are initially responsive to TGF-β-mediated inhibitory effects. 491 , 749 , 759 , 764 , 801 , 802 On the contrary, deficient TGF-β signaling can potently promote the growth of tumors. The downregulation of tumor TGF-β signaling in many cases is attributed to reduced expression or inactivating mutations of TβR or SMAD, as shown in various tumor types such as leukemia, 772 lymphoma, 803 , 804 esophageal cancer, 805 – 807 gastric cancer, 808 colorectal cancer, 30 , 807 , 809 – 811 pancreatic cancer, 32 , 812 , 813 biliary cancer, 812 ampullary cancer, 814 thyroid cancer, 815 prostate cancer, 816 , 817 breast cancer, 818 ovarian cancer, 819 endometrial cancer, 808 genital squamous cell carcinomas (SCC), 764 head and neck SCC, 820 – 823 etc. These changes are able to confer resistance to the tumor-inhibitory effects of TGF-β. In mouse models, tumors developed from tissues with deletion or inactivation of TβR exhibit increased cell proliferation and decreased cell apoptosis, accompanied by reduction in p15, p21, and p27, the elevation of MYC, cyclin D1, and epidermal growth factor receptor (EGFR), as well as activation of STAT3 and PI3K/AKT pathways. 761 – 765 Interestingly, reconstituted expression of TβRII in tumor cells with corresponding deficiency not only restores the inhibitory responses to TGF-β but also significantly attenuates the tumorigenicity of these cells. 824 Notably, TGF-β can fail to suppress the growth of tumors where there is likely no loss of functional TGF-β signaling components, and even formerly inhibited tumor cells can subsequently resume proliferating in vitro and develop larger tumor masses in vivo. 354 , 825 , 826 On one hand, such resistance may result from the dysfunction of the downstream targets of TGF-β signaling such as CKIs. 354 On the other hand, the tumor-suppressive signaling of TGF-β can be offset or interfered by enhanced I-SMAD activity 827 or potent oncogenic factors such as E1A, 828 , 829 EVI1, 148 , 830 SKI, 150 , 152 SNO, 153 MYC, 831 ID2, 832 mutant p53, 833 as well as RAS/RAF/ERK signaling. 162 , 834 Moreover, TGF-β-mediated tumor-promoting effects can also account for the enhanced tumor growth in vivo, as discussed in a later section. Contrary to its role as a suppressor of tumor growth, TGF-β generally acts as a promoter of tumor invasion and metastasis especially in advanced tumors. Upregulation of TGF-β as well as its receptors is associated with disease progression and poor prognosis in some patients with tumors such as breast cancer, 835 , 836 pancreatic cancer, 837 , 838 and gastric cancer. 839 Consistently, TGF-β overexpression or pre-treatment enables tumor cells to form increased metastases in vivo, 825 , 840 while loss of TGF-β responsiveness due to the introduction of dominant negative TβRII decreases the metastatic efficiency of high-grade tumor cells. 841 , 842 Moreover, tumors derived from transgenic murine epithelia that overexpress TGF-β or TβR are significantly more malignant and more invasive. 491 , 749 , 750 , 802 , 843 Notably, these TGF-β-overexpressing tumor cells are more likely to undergo the transition from epithelial cell phenotype into spindle cell phenotype which is the most malignant and invasive cell type. 802 , 843 This indicates that TGF-β can facilitate the progression of epithelial-derived tumors through the induction of EMT which is inoperative in tumors with deficiencies in TβR or SMAD. 761 , 770 , 774 , 842 , 843 Similar to the EMT of normal cells, TGF-β-induced EMT of tumor cells is characterized by changes in keratin, integrin, cadherin, catenin, claudin, vimentin, occludin, fibronectin, and MMP expression which can contribute to the invasive and metastatic capacity of tumors. 197 , 198 , 200 , 203 , 230 , 232 , 750 , 774 , 802 , 843 – 848 However, loss of functional TGF-β signaling components can occur in tumor cells during disease progression. 759 , 809 In fact, reduced TGF-β signaling can also contribute to tumor invasion and metastasis. For some patients, decreased expression of TβR is correlated with higher tumor grades, later clinical stages, and worse clinical prognosis. 805 , 816 , 818 A large number of cell models and mouse models also demonstrate that tumors lacking TGF-β signaling tend to be more malignant and more aggressive. 758 , 760 – 762 , 764 , 801 , 841 , 843 , 849 – 852 Relevant mechanisms in these cases involve the loss of E-cadherin, 761 the reduction in PAI, 849 the increase in RHO/RAC signaling, 843 the activation of integrin/focal adhesion kinase (FAK)/SRC/MAPK pathway, 764 and more importantly, the overexpression of various pro-invasive and pro-metastatic factors. In mouse models, deficient TGF-β signaling can stimulate tumor cells and stromal cells to produce high levels of TGF-β and other tumor-promoting factors such as CTGF, VEGF, IL-1β, C-X-C motif chemokine ligand (CXCL8), CXCL12, cyclooxygenase(COX)-2, MMPs, collagen, and tenascin C (TNC) which can strongly promote tumor angiogenesis, fibroblasts activation, immune infiltration, and ECM remodeling. 760 – 764 , 774 , 843 , 850 TGF-β can stimulate tumor progression even when its signaling pathways are unavailable in the tumor cells, indicating its additional tumor-promoting effects exerted on tumor stroma. 760 – 764 , 843 Fibroblasts, endothelial cells, and immune cells are the major stromal cell types in TME and can all be manipulated by TGF-β in favor of tumor progression. Actively produced TGF-β in the TME can stimulate the chemotactic migration of fibroblasts and convert them into MFs which are also known as cancer-associated fibroblasts (CAFs) in terms of tumors. 305 , 853 Activated CAFs can in turn repay TME with more TGF-β as well as other tumor-promoting factors such as TGF-α, FGF, HGF, PDGF, and CTGF to exert a strong stimulation on tumor growth. 324 , 853 – 857 Moreover, TGF-β regulates the production of various ECM components and remodelers by CAFs to facilitate the migration of tumor cells during invasion and metastasis. 855 , 858 Interestingly, fibroblasts with the loss of TβRII can also contribute to tumor development through the production of TGF-α, HGF, and macrophage-stimulating protein (MSP). 859 Endothelial cells can also be converted into CAFs through TGF-β-mediated EndMT. 860 More importantly, TGF-β promotes the angiogenesis of endothelial cells by inducing VEGF production in tumor cells and fibroblasts. 323 , 354 , 750 , 861 , 862 TGF-β also disrupts inter-endothelial junctions to increase the vascular permeability in TME through the process of EndMT and the induction of angiopoietin-like 4 (ANGPTL4). 863 Therefore, TGF-β-mediated angiogenesis not only increases the blood supply to tumors to favor their growth but also provides tumors with more accessible entrances into the circulation to form metastasis. Furthermore, TGF-β can modulate immune cell activity to facilitate tumor survival and development. TGF-β inhibits the tumoricidal activity of macrophages and neutrophils and polarizes them into tumor-promoting M2 macrophages and N2 neutrophils, which are also known as tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) in terms of tumors. 439 , 442 , 444 , 448 , 449 , 864 It also promotes the functions of Tregs while suppressing the cytotoxicity of CTLs and NK cells to facilitate tumor evasion from immune surveillance. 865 – 869 Moreover, TGF-β can inhibit the expression of MHC antigens in tumor cells to further attenuate their recognition by adaptive anti-tumor immunity. 870 , 871 However, TGF-β-mediated downregulation of MHC antigens and NKG2D ligands can increase tumor susceptibility to NK cytotoxicity to some extent. 233 , 872

TGF-β was first reported in 1978 when De Larco and Todaro discovered the ‘sarcoma growth factors’ which were produced by transformed murine fibroblasts and were able to transform normal fibroblasts to anchorage-independent growth. 1 In 1981, Roberts et al. successfully isolated and purified TGF-β from non-neoplastic murine tissues, 3 while at about the same time, Moses et al. independently accomplished the purification and characterization of the cytokine as well. 6 Both groups also noticed that this relatively acid- and heat-stable polypeptide required disulfide bonds for activity and was sensitive to disulfide-reducing agent dithiothreitol. In 1983, studies by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels indicated that the 25,000-dalton TGF-β molecule in humans was actually composed of two 12,500-dalton subunits cross-linked by disulfide bonds. 7 , 8 Two years later, the amino-acid sequence of human TGF-β1, the first known TGF-β isoform, was revealed by Derynck et al. through direct protein sequencing and complementary deoxyribonucleic acid (DNA) cloning. 2 The sequencing established that the 112-amino-acid-long TGF-β1 monomer is initially synthesized as the C-terminal segment of a 390-amino-acid-long precursor polypeptide. 2 By the time of 1988, researchers had realized that TGF-β generally remained non-covalently associated with the N-terminal segment of its precursor when it was secreted. 9 , 10 TGF-β cannot bind to its receptors with its receptor-binding site being masked in this inactive form, however, certain treatments such as acidification could convert latent TGF-β complex into active TGF-β ligand. 11 In addition, the other two TGF-β isoforms in mammals, TGF-β2 and TGF-β3, were respectively identified in 1987 12 and 1988. 13 , 14 Although the three TGF-β isoforms are encoded by three different genes, their mature ligands show strong conservation of amino acid sequences. The effects of TGF-β signaling in cell proliferation, 15 , 16 cell differentiation, 17 , 18 embryonic development, 19 wound healing, 20 immune regulation, 21 , 22 tissue fibrosis, 23 , 24 and tumor development 25 , 26 have been studied shortly after the discovery of the cytokine. Meanwhile, the receptors in TGF-β signaling known as TGF-β receptor I (TβRI) and TβRII were also identified and characterized in the 1980s. 27 – 29 But it was not until the discovery of signaling mediators small (Sma) in Caenorhabditis elegans and mothers against decapentaplegic (Mad) in Drosophila melanogaster that the homologous small mothers against decapentaplegic (SMAD) proteins were identified as the canonical signal transducers of TGF-β signaling in humans in 1996. 30 – 32 Since then, the development of TGF-β research has been largely accelerated. In recent times, as studies on TGF-β signaling in both health and disease going deeper and further, a lot of TGF-β-targeting therapies have been developed and assessed for the treatment of various diseases, 33 – 39 revealing a promising future for the studies in this area ( Fig. 1 ) . Fig. 1 History of research on TGF-β signaling History of research on TGF-β signaling

TGF-β signaling is so extensively and indispensably involved in a large number of biological processes that it has attracted great interest and attention over the past decades during which relevant knowledge has exploded in the fields of health, disease, and therapeutics. However, there are still some specific issues that have not been fully elucidated, while some previous knowledge is facing updates and challenges. Studies on embryonic development and wound healing have revealed the isoform-specific roles of TGF-β which remain poorly aware in other fields of research, as studies on immune homeostasis, fibrotic diseases, and tumor development so far have focused on the most abundant TGF-β1 isoform in particular. Since all TGF-β isoforms are believed to signal through the same receptors and downstream pathways, the causes of the differences in biological effects between isotypes have not been fully understood. Moreover, since a natural TGF-β heterodimer containing one TGF-β1 monomer and one TGF-β2 monomer has long been discovered, 12 , 1068 it would be very interesting to identify and characterize novel TGF-β heterodimers in the future. Furthermore, with the discovery and study of TGF-β superfamily which also includes polypeptides structurally similar to TGF-β such as nodal, myostatin, inhibins, activins, Müllerian-inhibiting substance (MIS), bone morphogenetic proteins (BMPs), and growth and differentiation factors (GDFs), researchers have realized that TGF-β can also signal through pathways ‘specific’ to other TGF-β superfamily members, for example, via receptors ALK1/2/3 and transcription factors SMAD1/5/8. 1069 – 1073 The significance of the signaling crosstalk within the TGF-β superfamily also warrants future exploration. Notably, Reblozyl (luspatercept or ACE-536), a ligand trap that contains the extracellular domain of human activin receptor type IIB (ActRIIB) to inhibit GDF11-mediated SMAD2/3 signaling has been approved by the US Federal Drug Agency (FDA) for the treatment of anemia in adult patients with β-thalassemia or with MDS. As for TGF-β-targeting therapy, the efficacy and safety of treatment are always issues of concern. The current lack of systematic studies on the dural roles of TGF-β in wound healing, infectious diseases, and tumor development may hinder the development of related therapeutics. Given the extensive impacts of TGF-β on a lot of biological processes, the development of TGF-β isoform-specific therapies and SMAD-binding peptide aptamers is expected to cause less adverse effects through more precise targeting. Moreover, the identification of the applicable population for each therapeutic approach is also important for better efficacy and less toxicity. Serum and tissue levels of TGF-β have shown potential as predictors or indicators of the development, 1074 – 1077 complication, 1078 – 1080 response, 1081 – 1084 recurrence, 1085 – 1087 and outcomes 1088 – 1090 of various kinds of diseases, meanwhile, bioinformatic tools of TGF-β signaling-related gene expression signatures have also been developed for patient stratification. 863 , 1091 But so far, TGF-β or related factors as clinical biomarkers still need further development and assessment. To summarize, this review focuses on the multiple roles of TGF-β in health and disease while emphasizing the mechanisms of TGF-β production, activation, signaling, as well as corresponding therapeutic strategies. These understandings might be instructive for the basic and applied research of relevant topics in the future.

During the biosynthesis of TGF-β, the precursor undergoes post-translational processing to become a latent complex which is the secretory form of TGF-β. The latent TGF-β complex still requires further activation to eventually become a mature cytokine before it can trigger signal transduction in cells (Fig. 2 ). Fig. 2 Biosynthesis and activation of TGF-β. Each TGF-β monomer is initially synthesized as a precursor polypeptide. In the endoplasmic reticulum, TGF-β precursors lose their signal peptides and dimerize through disulfide bonds. The dimers then transit into the Golgi where they are cleaved by protease furin into mature cytokine segments and latency-associated peptides (LAPs) to form small latent complexes (SLCs). The secreted SLCs can further link to latent TGF-β-binding proteins (LTBPs) which target them into the extracellular matrix (ECM) for storage, or they can link to glycoprotein-A repetitions predominant protein (GARP) or leucine-rich repeat-containing protein 33 (LRRC33) which tethers them to the cell surface. Numerous factors such as acids, bases, reactive oxygen species (ROS), thrombospondin-1 (TSP-1), certain proteases, and integrins can release the mature cytokines from the latent complexes and thus are known as TGF-β activators Biosynthesis and activation of TGF-β. Each TGF-β monomer is initially synthesized as a precursor polypeptide. In the endoplasmic reticulum, TGF-β precursors lose their signal peptides and dimerize through disulfide bonds. The dimers then transit into the Golgi where they are cleaved by protease furin into mature cytokine segments and latency-associated peptides (LAPs) to form small latent complexes (SLCs). The secreted SLCs can further link to latent TGF-β-binding proteins (LTBPs) which target them into the extracellular matrix (ECM) for storage, or they can link to glycoprotein-A repetitions predominant protein (GARP) or leucine-rich repeat-containing protein 33 (LRRC33) which tethers them to the cell surface. Numerous factors such as acids, bases, reactive oxygen species (ROS), thrombospondin-1 (TSP-1), certain proteases, and integrins can release the mature cytokines from the latent complexes and thus are known as TGF-β activators Each TGF-β monomer is initially synthesized as a precursor polypeptide composed of a mature cytokine as its C-terminal segment, a signal peptide at the N-terminus, and a latency-associated peptide (LAP) in between. 2 The signal peptide leads the precursor into the endoplasmic reticulum lumen and promptly gets removed. The remainder of the precursor then dimerizes through three disulfide bonds and transits into the Golgi where it gets cleaved between the mature cytokine and LAP by protease furin. 40 However, the cytokine segment is still unable to bind its receptors after the cleavage, for it remains associated with LAP in a non-covalent way that masks its receptor-binding site and forms a small latent complex (SLC). 41 In most cases, LAP is linked to latent TGF-β-binding protein (LTBP) through a disulfide bond, making the SLC into a large latent complex (LLC) when secreted. 42 LTBP can further bind to fibrillin to target the LLC into the extracellular matrix (ECM) for storage. 43 Alternatively, LAP can also form disulfide linkage with leucine-rich repeat-containing protein 32 (LRRC32) or LRRC33 to tether SLC to the cell surface. Unlike LTBP which is widely expressed by many cell types, LRRC32, also known as glycoprotein-A repetitions predominant protein (GARP), is specifically detected in regulatory T cells (Tregs), platelets, and endothelium, 44 whereas high expression of LRRC33 is found in macrophages, dendritic cells (DCs), and B cells. 45 The bioactivity of TGF-β is based on ligand-receptor interaction which requires the exposure of its receptor-binding site. Thus, the activation of TGF-β represents the release of mature cytokine from the latent complex. Numerous factors have been identified as TGF-β activators as introduced below. Notably, integrin-dependent activation is so far the best described and likely the most important mechanism, while TGF-β activation mediated by acids, bases, reactive oxygen species (ROS), thrombospondin-1 (TSP-1), proteases, and other TGF-β activators is collectively known as integrin-independent activation. Integrins are heterodimeric transmembrane receptors each consisting of an α-subunit and a β-subunit. TGF-β activation by integrins requires the binding of the integrins to an RGD sequence in the LAP of TGF-β1 and TGF-β3. Therefore, latent TGF-β2 without the RGD motif is excluded from integrin-dependent activation. 46 Among all integrins, αVβ6 and αVβ8 integrins are the best studied TGF-β activators. The expression of αVβ6 integrin is nearly restricted to epithelial cells and is upregulated in response to morphogenesis, wounding, inflammation, and tumorigenesis. 47 In contrast, αVβ8 integrin is widely expressed by epithelial cells, 48 fibroblasts, 49 macrophages, 50 DCs, 51 Tregs, 52 and different kinds of tumor cells. 53 The lack of αVβ6 and αVβ8 integrin activity reproduces the phenotypes of TGF-β1- and TGF-β3-null mice, indicating the central importance of integrin-dependent activation. 54 , 55 Upon binding to the RGD motif in LAP, the mechanisms by which αVβ6 and αVβ8 integrins activate TGF-β are quite different. With latent TGF-β being tethered to ECM or cell membrane (through the binding of LAP to LTBP, GARP, or LRRC33 as mentioned before) and the cytoplasmic domain of integrin β6 subunit linking to the actin cytoskeleton, αVβ6 integrin can transmit contractile force which changes the conformation of LAP to release TGF-β ligand. 56 , 57 However, the cytoplasmic domain of integrin β8 subunit does not link to the actin cytoskeleton. One effective mechanism for αVβ8 integrin-mediated TGF-β activation requires the proteolytic activity of membrane type 1-matrix metalloproteinase (MT1-MMP, also known as MMP14). 48 Alternatively, membrane molecules such as GARP and LRRC33 which bind and present latent TGF-β on the surface of one cell can cooperate with the αVβ8 integrin expressed on a different cell to activate TGF-β in trans. 45 , 58 , 59 A recent study reveals that upon binding to αVβ8 integrin, the flexible membrane-presented latent complex can expose the active domain of the TGF-β ligand to its receptors for binding and signaling without the need to release diffusible cytokine. 60 It has long been noticed that acidification can unmask the activity of freshly secreted TGF-β. 61 Sharply defined parameters for human TGF-β activation by acids and bases show that the transition from latency of all three isoforms occurred between pH 2.5 and 4, and between pH 10 and 12. 62 Thus, extremely acidic environments such as the microenvironments in tumor tissues and the resorption lacunae of osteoclasts are possibly conducive to local TGF-β activation. 63 , 64 A study on lung fibrosis even suggests that physiologic concentrations of lactic acid are sufficient enough to activate TGF-β in a pH-dependent manner. 65 TGF-β1 is the only isoform that can be directly activated by ROS, for a unique methionine residue at the amino acid position 253 of its LAP is required for oxidation-triggered conformational change. 66 However, ROS can induce other TGF-β activators such as TSP-1 67 and MMPs 68 to activate all three isoforms in an indirect manner. ROS-mediated TGF-β activation prevails in tissues exposed to asbestos, 69 , 70 ultraviolet, 68 and ionizing radiation. 71 High glucose intake can also induce ROS production and consequentially increase TGF-β activation to play roles in the development of fibrotic diseases and inflammatory diseases. 72 , 73 Moreover, in T cells, ROS can be elevated during apoptosis or upon stimulation by T cell receptor (TCR) and cluster of differentiation 28 (CD28) to contribute to the immunosuppression mediated by activated TGF-β. 74 , 75 TSP-1 is a multi-functional ECM protein not only abundant in platelet α-granules but also secreted by fibroblasts, endothelial cells, macrophages, T cells, and many other cell types. 76 The KRFK sequence in TSP-1 can recognize the LSKL sequence in LAP to competitively disrupt its interaction with the receptor-binding site of the TGF-β ligand. Since the LSKL sequence in LAP is conserved among TGF-β isoforms, it is suggested that the direct binding of TSP-1 to latent complex is capable of activating all three TGF-β isoforms through this protease- and cell-independent mechanism. 77 Interestingly, TSP-1 can also bind to the mature TGF-β ligand to form a complex that retains the biological activity of the cytokine. 78 ROS, 67 glucose, 79 angiotensin II, 80 hypoxia, 81 wounding, 82 inflammation, 83 pathogens, 84 – 86 and many other factors can all induce TSP-1 to function as a TGF-β activator in wound healing, 67 , 82 cardiovascular diseases, 81 , 86 renal diseases, 79 fibrotic diseases, 87 , 88 inflammatory diseases, 83 infectious diseases, 89 and tumors. 90 Many proteases have been proved capable of directly activating TGF-β in vitro. However, the function of an individual protease seems redundant in vivo, as deficiency of a single species generally leads to no significant signs of impaired TGF-β activation. 91 Among these proteases, MMPs such as MMP-2, MMP-9, and MMP-13 are conducive to the TGF-β activation in wound healing, 92 cardiovascular diseases, 93 renal diseases, 94 fibrotic diseases, 95 and tumors. 96 Interestingly, although the activation by MMPs works for all three TGF-β isoforms, latent TGF-β2 and TGF-β3 appear much more sensitive to MMP-9 treatment than latent TGF-β1. 96 Moreover, a serine protease known as plasmin plays an important role in the TGF-β activation mediated by macrophages 97 , 98 and endothelial cells. 99 , 100

The studies on TGF-β started as early as the 1980s and have developed rapidly ever since. Although TGF-β was first found to be secreted by transformed cells, 1 it is widely produced by non-neoplastic tissues such as salivary glands, muscles, kidneys, liver, heart, brain, and embryos as well. 2 – 4 In fact, platelets have been identified as one of the most abundant sources of TGF-β among all normal tissues. 5 The ubiquitous expression of TGF-β in health strongly indicates its critical and multiple roles in physiological conditions. Accumulating evidence has suggested that TGF-β functions diversely among different cell types in a context-dependent manner. Generally, cell survival, metabolism, growth, proliferation, differentiation, adhesion, migration, and death are all under the regulation of TGF-β. Proper TGF-β signaling is critical to the normal functioning and homeostasis of healthy bodies while aberrant TGF-β signaling can lead to diseases of various categories. For this reason, numerous targeted therapies that can remedy dysregulated TGF-β activity have been developed with some demonstrating encouraging safety and efficacy in clinical trials. In this review, we focus on the mechanism, physiology, pathology, as well as therapeutics of TGF-β signaling, aiming to provide historical, current, and future perspectives on relevant topics.

To rectify the dysfunction of TGF-β in different kinds of diseases, several targeted therapies have been developed to regulate TGF-β activity at the levels of biosynthesis, activation, and signaling. Many completed clinical trials have preliminarily confirmed the safety and efficacy of some therapeutic strategies, while there are still numerous clinical trials ongoing at present (Table 1 ). Table 1 Ongoing clinical trials of TGF-β-targeting therapies Targets Strategies Treatments Diseases Clinical trials TGF-β2 mRNA Antisense oligonucleotide Trabedersen Pancreatic ductal adenocarcinoma and malignant pleural mesothelioma NCT06079346 (phase 2/3) and NCT05425576 (phase 2) TGF-β2 mRNA Antisense oligonucleotide TASO-001 Solid tumors NCT04862767 (phase 1) Furin ShRNA Vigil Ovarian cancer NCT02346747 (phase 2) Latent TGF-β complex Monoclonal antibody SRK-181 Solid tumors NCT04291079 (phase 1) GARP Monoclonal antibody HLX60 Solid tumors and lymphoma NCT05483530 (phase 1) and NCT05606380 (phase 1) TGF-β2 Dietary supplement Modulen IBD NCT04921033 (phase 3), NCT04777656 (phase 3), and RBR-955md27 TGF-β Monoclonal antibody NIS793 Pancreatic cancer, colorectal cancer, and MDS NCT04935359 (phase 3), NCT04390763 (phase 2), NCT04952753 (phase 2), NCT05417386 (phase 1), and NCT04810611 (phase 1) TGF-β Monoclonal antibody SAR439459 Multiple myeloma and osteogenesis imperfecta NCT04643002 (phase 1/2) and NCT05231668 (phase 1) TGF-β Ligand trap Bintrafusp alfa Solid tumors NCT05061823 (phase 3), NCT03436563 (phase 2), NCT04396886 (phase 2), NCT05005429 (phase 2), NCT04708470 (phase 1/2), NCT04574583 (phase 1/2), etc. TβR Dominant-negative TβR TGF-β-resistant cytotoxic T lymphocytes Lymphoma NCT00368082 (phase 1) TβRI Kinase inhibitor Galunisertib Nasopharyngeal carcinoma, prostate cancer, colorectal cancer, and glioma NCT04605562 (phase 2), NCT02452008 (phase 2), NCT02688712 (phase 2), NCT01582269 (phase 2), NCT05700656 (phase 1/2), and NCT01682187 (phase 1) TβRI Kinase inhibitor Vactosertib Solid tumors and myeloproliferative neoplasm NCT04515979 (phase 2), NCT04064190 (phase 2), NCT05436990 (phase 2), NCT04103645 (phase 2), NCT05588648 (phase 1/2), NCT03802084 (phase 1/2), etc. TβRI Kinase inhibitor LY3200882 Solid tumors NCT02937272 (phase 1) Ongoing clinical trials of TGF-β-targeting therapies Trabedersen (AP 12009 or OT-101) is an antisense oligonucleotide complementary to human TGF-β2 mRNA and can specifically inhibit TGF-β2 biosynthesis. It is hypothesized that trabedersen mainly acts by reversing TGF-β2-mediated immunosuppression to facilitate immune responses against tumors. A phase 2b clinical trial showed no advantage in early tumor control rate but in long-term survival rate for glioma patients treated with trabedersen in comparison with standard chemotherapy. Tumor responses which continued to increase long after discontinuation in the study suggested that the clinically relevant beneficial effects of trabedersen might increase over time. Moreover, compared with the standard chemotherapy group, drug-related or possibly drug-related adverse events in the trabedersen group were less common and mostly nervous system disorders. The study also indicated that the optimal dose of trabedersen is 10 µM, as both its efficacy and safety tended to be superior to the 80 µM dose, although the mechanism for this counterintuitive result has not been fully understood. 873 TGF-β1 antisense oligonucleotides or small interfering RNAs (siRNAs) were also developed and evaluated in different pre-clinical models, suggested as potential therapeutic strategies for tuberculosis, 874 , 875 wound scarring, 876 , 877 and several renal diseases. 878 – 881 TGF-β antisense gene-modified tumor cell vaccines are designed to exhibit increased immunogenicity due to reduced TGF-β expression in the tumor cells that comprise the vaccines. Vaccine Lucanix (belagenpumatucel-L) made from allogeneic non-small cell lung cancer (NSCLC) cell lines was well tolerated and brought survival advantages to NSCLC patients who were randomized within 12 weeks of completion of platinum-based chemotherapy and in those who had received prior radiation, as shown in a phase 3 trial which, however, failed to demonstrate a significant increase in survival in the overall patient population. 882 TGF-β antisense-modified autologous tumor cell vaccines have also been tested in advanced glioma and other solid tumors, respectively, in two phase 1 studies in which enhanced anti-tumor activity and improved survival were observed. 34 , 883 Notably, in the study among glioma patients, the most common treatment-related adverse events were delayed-type hypersensitivity-like reactions observed at the sites of the second and subsequent vaccinations in all patients. Some of these patients also experienced transient, flu-like symptoms consisting of musculoskeletal aches and pains and fatigue during the course of treatment. 34 Convertase furin is a therapeutic target participating in the post-translational processing of TGF-β. Vigil (FANG or Gemogenovatucel-T) is an autologous tumor cell vaccine incorporating a plasmid encoding granulocyte-macrophage colony-stimulating factor (GMCSF) and a bifunctional short-hairpin RNA (shRNA) targeting the expression of furin. A phase 1 study confirmed its safety and efficacy in various advanced solid tumors, with significant survival differences noted between patients who received less than four vaccinations and those who received no less than four vaccinations. 884 A later phase 2b trial also demonstrated significant clinical benefit in homologous recombination proficient ovarian cancer ( NCT02346747 ). 885 Both studies reported no treatment-related serious adverse events, while the most common grade one and two adverse events related to study medication were local reactions at the injection site. SRK-181 is an antibody that selectively binds to latent TGF-β1 to inhibit its activation. Co-administration of SRK-181 and anti-PD-1 antibody induced profound anti-tumor responses and survival benefit in mice, with increased infiltrating CD8+ T cells and decreased immunosuppressive myeloid cells observed in tumors refractory to anti-PD-1 treatment. 886 The selective blockade of TGF-β1 by SRK-181 neither caused cardiac valvulopathy in rats as pan-TGFβ inhibitors might do nor did it induce cytokine release in human peripheral blood. Moreover, SRK-181 showed no effect on human platelet aggregation, activation, and binding. 886 , 887 The favorable safety profile displayed in these preclinical assessments supports the ongoing phase 1 trial of SRK-181 in patients with advanced cancers ( NCT04291079 ). GARP expressed by Tregs, platelets, and endothelium functions to tether latent TGF-β complex to the cell surface for activation. Anti-GARP monoclonal antibody PIIO-1 proved to be an effective and safe strategy to block TGF-β activation in preclinical models, for it specifically bound to ligand-free GARP on Tregs but lacked recognition of GARP-latent TGF-β complex on platelets, actually avoiding the risk of platelet-related toxicities such as thrombocytopenia. More importantly, PIIO-1 showed therapeutic efficacy against both GARP+ and GARP- cancers alone or in combination with anti-PD-1 antibody, by preventing T cell exhaustion and enhancing CD8+ T cell migration into the TME in a C-X-C motif chemokine receptor 3 (CXCR3)-dependent manner. 888 Integrins are regarded as the most important activators of TGF-β. Abituzumab (EMD 525797 or DI17E6) is an antibody against pan-αV integrins. In a phase 1/2 trial on KRAS wild-type metastatic colorectal cancer ( NCT01008475 ), the progression-free survival (PFS) and response rates were similar among all groups in the intent-to-treat population comprising all patients randomized, although a trend toward improved overall survival (OS) was observed in the groups that received abituzumab treatment. However, exploratory analysis suggested that in patients with high αVβ6 expression, PFS and response rates might be increased with abituzumab therapy. 889 This pan-αV integrin inhibitor was also found to inhibit prostate cancer-associated bone lesion formation in a randomized phase 2 trial ( NCT01360840 ), although PFS was not significantly extended. 890 Recently, abituzumab has been investigated in SSc-associated interstitial lung disease in a phase 2 trial ( NCT02745145 ). However, the study was terminated prematurely due to slow enrollment and no meaningful conclusions could be drawn due to a small sample size. 891 The most commonly reported treatment-related adverse events of abituzumab included fatigue, headache, gastrointestinal disorders, as well as abnormal biochemistry and hematology values. 889 , 890 , 892 Cilengitide (EMD 121974, NSC 707544) is a selective αvβ3 and αvβ5 integrin inhibitor which has been evaluated for therapeutic efficacy in NSCLC ( NCT00842712 ), 893 , 894 head and neck SCC ( NCT00705016 ), 895 glioblastoma ( NCT00689221 , NCT00813943 , and NCT01124240 ), 896 – 903 melanoma, 904 pancreatic cancer, 905 and prostate cancer 906 , 907 in a series of phase 2 studies and one phase 3 study. Although cilengitide failed to demonstrate significant clinical benefits in these studies on tumors, it might be a novel treatment for fibrotic diseases as relevant preclinical studies suggested. 908 , 909 Notably, the adverse events possibly related to cilengitide treatment included fatigue, arthralgia, lymphopenia, and gastrointestinal disorders. 893 , 897 , 899 , 900 , 904 , 906 , 907 Furthermore, an inhibitor of pan-integrins and TGF-β known as GLPG-0187 was proved to enhance T cell killing of colorectal cancer cells in vitro, possibly by suppressing TGF-β-mediated PD-L1 upregulation. 910 , 911 TSP-1 can directly activate all three TGF-β isoforms independent of other activators or cellular activity. The conserved LSKL sequence in LAP which is recognized by TSP-1 can be synthesized as peptides to block TSP-1-mediated TGF-β activation. Pre-clinical studies suggested that treatment of LSKL or relevant tripeptide SRI31277 could be novel therapeutic strategies for various cardiovascular diseases, 912 pulmonary diseases, 913 renal diseases, 914 – 916 nervous diseases, 917 , 918 fibrotic diseases, 919 – 921 wound healing, 922 , 923 and tumors. 924 – 926 Moreover, TSP-1 antisense oligonucleotides were successfully developed and applied to inhibit TGF-β activation in a rat model of mesangial proliferative glomerulonephritis, demonstrating a remarkable prevention against renal fibrosis. 927 A TGF-β2-enriched polymeric dietary supplement known as Modulen (CT3211) was effective in inducing earlier remission of inflammatory bowel diseases (IBDs) including both Crohn’s disease and UC with significant improvements in endoscopic and histologic appearances, mucosal cytokine parameters, C-reactive protein (CRP) values, erythrocyte sedimentation rates (ESRs), serum albumin levels, as well as weight and height scores in the patients. 928 – 931 Notably, an exclusive Modulen diet was more efficient than steroids to induce mucosal healing in children with Crohn’s disease, possibly due to its additional advantage in regulating intestinal microbiota ( NCT00265772 ). 932 , 933 Moreover, a pre-operative polymeric diet enriched with TGF-β2 was able to decrease post-operative complications after surgery for complicated ileocolonic Crohn’s disease. 934 The side effects of Modulen were mild, including abdominal pain, flatulence, nausea, and vomiting. 928 , 932 , 934 In mouse models, oral TGF-β supplementation also showed beneficial effects on food allergy prevention. 935 – 937 In fact, it is believed that the presence of TGF-β in breast milk can protect the progeny from several allergic diseases such as asthma, 938 eczema, 939 and food allergy. 940 Recombinant human TGF-β3 known as avotermin (Juvista) is a potential therapy for the improvement of cutaneous scarring. In a series of phase 1/2 studies ( NCT00847925 , NCT00847795 , NCT00629811 , NCT00432211 , NCT00594581 , and NCT00430326 ), visual assessment of scar formation revealed that, in contrast to placebo, intradermal avotermin could significantly improve total scar scores which were derived from a visual analog scale to assess how closely scars resembled normal skin. The results were further confirmed by histological assessments that scars treated with avotermin showed better organized ECM of the papillary and reticular dermis. The incidence of adverse events at wound sites, including infection, exudate, erythema, pain, burning, itching, and thickening was low and similar for avotermin and controls. 941 – 944 Although the other two TGF-β isoforms, TGF-β1 and TGF-β2, showed no therapeutic activity of scarring, they were found to improve and accelerate the healing of cutaneous wounds in animal models as well as clinical patients. 304 , 306 , 307 , 317 , 321 , 334 , 945 Moreover, TGF-β also showed therapeutic potential for tissue regeneration, 329 , 946 , 947 inflammatory diseases, 676 , 687 , 948 and influenza 949 as shown in relevant preclinical models. TGF-β neutralizing antibodies and ligand traps can block the binding of TGF-β to its receptors. Fresolimumab (GC1008), a monoclonal antibody that neutralizes all three TGF-β isoforms demonstrated acceptable safety and preliminary evidence of anti-tumor activity in a phase 1 study on advanced malignant melanoma and renal cell carcinoma ( NCT00356460 ). 950 In a phase 2 trial ( NCT01401062 ), a higher dose of fresolimumab is associated with longer median OS as well as improved peripheral blood mononuclear cell counts and boosted central memory CD8+ T cell levels in metastatic breast cancer patients receiving radiotherapy. 951 Fresolimumab also showed therapeutic effects on SSc with decreased biomarkers of skin fibrosis and improved clinical symptoms in the patients in a phase 1 study ( NCT01284322 ). 952 Moreover, a phase 1 study evaluated the safety of fresolimumab in patients with treatment-resistant primary focal segmental glomerulosclerosis and the good tolerability supported additional evaluation in larger randomized dose-ranging clinical trials. 953 Notably, the major drug-related adverse events of fresolimumab were skin disorders, bleeding episodes, and anemia. Skin toxicity was particularly significant and tumor patients assigned to high doses of treatment even developed skin tumors, including keratoacanthoma, basal cell carcinoma, and SCC. 950 – 954 Another anti-TGF-β monoclonal antibody known as NIS793 was well tolerated alone or in combination with anti-PD-1 antibody in patients with advanced solid tumors in a phase 1 study ( NCT02947165 ). Treatment-related adverse events of all patients in the study were mostly skin toxicity and gastrointestinal events, and no dose-limiting toxicities were observed during dose escalation. Notably, biomarker analyses in the study showed evidence of systemic target engagement, local signaling inhibition, and tumor immune activation. 955 Apart from tumors, a recombinant human anti-TGF-β1 antibody known as CAT-192 was evaluated in the treatment of early-stage diffuse cutaneous SSc but showed no evidence of efficacy in the pilot phase 1/2 study. The most commonly reported adverse events in the study affected the gastrointestinal, musculoskeletal, respiratory, and skin systems, but none of them were considered to be related to the treatment. 956 Moreover, a phase 2 study assessing the safety and efficacy of TGF-β1 monoclonal antibody in patients with diabetic nephropathy was terminated early for futility ( NCT01113801 ). The frequencies of the various categories of adverse effects in this study were generally similar between the treatment and placebo groups. 957 Furthermore, monotherapy of a selective TGF-β1/3 trap known as AVID200 in a population of patients with an advanced stage of myelofibrosis in a phase 1b trial resulted in limited toxicity as well as improvements in spleen size, symptom benefit, and platelet counts ( NCT03895112 ). Remarkably, platelet count increase was a therapeutic effect not observed with other myelofibrosis therapies, suggesting a potential advantage of AVID200 treatment. Adverse events that occurred during the study regardless of attribution mainly included pruritus, fatigue, abdominal pain, anemia, and thrombocytopenia. 958 Additionally, other potential applications of neutralizing TGF-β antibodies suggested by pre-clinical studies include wound healing, 334 , 959 , 960 prostatic hyperplasia, 961 pulmonary diseases, 962 , 963 cardiovascular diseases, 564 , 964 musculoskeletal diseases, 965 – 968 inflammatory diseases, 969 , 970 and Chagas disease ( Trypanosoma cruzi infection). 971 Bifunctional antibody-ligand traps containing the extracellular domain of TβRII can target both TGF-β and immune checkpoints. In preclinical studies, both the anti-CTL associated protein (CTLA)-4-TβRII chimera and the anti-PD-L1-TβRII chimera exhibited superior anti-tumor efficacy compared with their parent immune checkpoint inhibitors. 972 Bintrafusp alfa (M7824), a bifunctional fusion protein targeting both TGF-β and PD-L1 was assessed in several phase 1 trials ( NCT02699515 , NCT02517398 , NCT02699515 , and NCT04247282 ). The results showed that bintrafusp alfa had encouraging efficacy in NSCLC, 973 gastric cancer, 974 biliary tract cancer, 975 as well as human papillomavirus (HPV)-unrelated head and neck cancer in which enhanced tumor antigen-specific immunity has been observed. 976 Similar to fresolimumab, the treatment-related adverse events of bintrafusp alfa included fatigue, colitis, bleeding, anemia, hypokalemia, lipase increase, hepatic function abnormalities, as well as several skin disorders from rash, hyperkeratosis, to keratoacanthoma and SCC. 973 – 977 BR102 is another bifunctional fusion protein simultaneously targeting PD-L1 and TGF-β. The efficacy and safety of BR102 demonstrated in preclinical characterization supported its further clinical development for anti-cancer therapy. 978 Notably, the bifunctional antibody-ligand traps have inspired the development of chimeric antigen receptor (CAR)-T cells secreting bispecific trap protein, which co-targets PD-1 and TGF-β to enhance anti-tumor efficacy as shown in mouse models. 979 Furthermore, LAP, TβRIII (β-glycan), and decorin can bind to TGF-β as natural inhibitors. They have shown treatment effects in preclinical models of wound healing, 980 – 983 cardiovascular diseases, 984 – 989 nervous diseases, 990 – 992 renal diseases, 993 – 996 fibrotic diseases, 997 – 1000 tuberculosis, 1001 and tumors 1002 – 1005 and thus warrant further development. TGF-β-insensitive CAR-T cells armored with dominant-negative TβRII showed preliminary evidence for early anti-tumor function in prostate cancer, including a biomarker decline among approximately 30% of the patients in a phase 1 trial ( NCT03089203 ). This strategy which is considered generally feasible, despite no partial response being observed in the study, and safe, with study-related serious adverse events mostly being cytokine release syndrome, warrants further validation and investigation. 1006 Dominant-negative TβRII can also enhance the anti-tumor efficacy of DC vaccines, manifested by powerful tumor-specific CTL responses, inhibited tumor development, and prolonged survival times in mouse models. 1007 , 1008 Moreover, dominant-negative TβRII showed great potential for reducing hypertrophic scars as in rabbit ear models. 1009 Many small-molecule inhibitors have been developed to suppress the kinase activity of TβRI. In a series of phase 2 studies, a TβRI kinase inhibitor known as galunisertib (LY2157299) showed preliminary efficacy in patients with myelodysplastic syndromes (MDS) ( NCT02008318 ), 1010 NSCLC ( NCT02423343 ), 1011 hepatocellular carcinoma ( NCT01246986 ), 1012 , 1013 rectal cancer ( NCT02688712 ), 1014 and pancreatic cancer, 1015 but failed to demonstrate clinical benefit in patients with glioma ( NCT01582269 and NCT01220271 ). 1016 , 1017 The most common adverse events related to galunisertib treatment included fatigue, pyrexia, anemia, nausea, vomiting, diarrhea, and abdominal pain. 1010 , 1013 , 1017 Despite comprehensive cardiovascular monitoring for galunisertib did not detect medically relevant cardiac toxicity in cancer patients, 1018 galunisertib-related uncontrolled cytokine release was reported in patients with advanced solid tumors in a phase 1 trial ( NCT01646203 ). 1019 Other TβRI kinase inhibitors such as SM16, SD-208, NP-40208, SB-431542, LY3200882, LY364947 , and vactosertib (EW-7197) also showed therapeutic potential in pre-clinical studies on tumors 1020 – 1026 as well as many other diseases such as cardiovascular diseases, 565 , 1027 – 1030 renal diseases, 1031 ophthalmic diseases, 1032 skeletal diseases, 1033 fibrotic diseases, 1034 – 1036 inflammatory diseases, 1037 – 1039 Chagas disease, 1040 , 1041 coronavirus disease 2019 (COVID-19), 1042 and wound healing. 1043 – 1045 An oral SMAD7 antisense oligonucleotide known as mongersen (GED-0301) showed promising results in patients with active Crohn’s disease in phase 1 and 2 phase trials, but further phase 3 study failed due to lack of clinical benefit (EudraCT 2009-012465-66, EudraCT 2011-002640-27, and NCT02596893 ). 1046 – 1048 Meanwhile, SMAD3 antisense oligonucleotide treatment was found to improve flexor tendon repair in mice and might have possible therapeutic applications in clinical practice. 877 Moreover, a small-molecule SMAD3 inhibitor known as specific inhibitor of SMAD3 (SIS3) has shown pre-clinical therapeutic efficacy in wound healing, 1049 cardiovascular diseases, 569 , 1050 , 1051 nervous diseases, 1052 renal diseases, 1053 , 1054 skeletal diseases, 1055 fibrotic diseases, 1056 , 1057 inflammatory diseases, 1039 , 1058 type 2 diabetes, 1059 , 1060 and tumors, 1061 , 1062 suggesting a novel approach that could be further tested to treat clinical patients. Furthermore, several SMAD-binding peptide aptamers have been developed to selectively inhibit the binding between SMADs and their interacting factors. 1063 An aptamer containing the SMAD-binding domain of transcription factor lymphoid enhancer-binding factor 1 (LEF1) can suppress tumor cell proliferation by inhibiting the interaction between SMAD4 and LEF/T cell-specific factor (TCF) to suppress MYC expression. 1064 Other aptamers that bind specifically to R-SMADs through the SMAD-binding domain from SARA can impair the formation of functional SMAD oligomers to inhibit TGF-β-induced EMT. 1065 , 1066 Moreover, aptamers that disrupt the interaction between SMAD and transcription coactivator yes-associated protein (YAP) have been designed for bone tumor therapy. 1067