Emerging roles of steroid receptor coactivators in stromal cell responses.

The SRC proteins are ubiquitously expressed in multiple cell types of the tissue stroma such as fibroblasts, endothelial and immune cells and all three SRCs regulate cell-autonomous and paracrine effects that influence stromal-epithelial cell interactions. The vasoprotective function of estrogen ( Orshal & Khalil 2004 ) is demonstrated by reduced neointima formation and inhibition of neointimal cell proliferation after carotid artery ligation in mice. Estrogen signals through ER complexed with SRCs. The SRC-1 protein is expressed in endothelial and vascular smooth muscle cells in mice. SRC-1 knockout mice that have been ovariectomized show increased neointimal formation after carotid artery ligation with estrogen treatment compared to wildtype mice ( Yuan & Xu 2007 ). Additionally, SRC-1 knockout mice display increased proliferation of endothelial, vascular smooth muscle and neointimal cells compared to wildtype mice. Decreased SRC-1 expression also reduces endothelial cell migration and tube formation in vitro ( Zhang & Shi 2019 ). Hence, SRC-1 function in endothelial and vascular smooth muscle cells is important for the vasoprotective function of estrogen. SRC-1 also supports the steroid hormone response in endometrial stromal cell decidualization ( Szwarc et al. 2014 ). Estrogen and progesterone regulate the ability of the uterus to support pregnancy. Before implantation, progesterone stimulates endometrial stromal cell proliferation. At the time of implantation, endometrial stromal cells undergo differentiation to form decidual cells that are in direct contact with the embryo. The decidual response can be stimulated in mice by treating ovariectomized mice with a high dose of progesterone and a low dose of estrogen followed by mechanical traumatization (scratch). SRC-1 knockout mice in this model show only a partial response to decidual stimulation compared to wildtype mice ( Xu et al. 1998 ). Additionally, endometrial stromal cells also express a proteolytic cleaved isoform of SRC-1 that is produced as a result of increased MMP9 activity during endometriosis ( Han et al. 2012 ). This cleaved SRC-1 isoform is present in the cytoplasm and nucleus and plays a role in protecting endometrial epithelial cell apoptosis in the absence of paracrine endometrial stromal cell signaling. SRC-2 also regulates the endometrial stromal cell decidualization process in response to steroid hormone stimulation. Like SRC-1 knockout mice, SRC-2 knockout mice show a significant reduction in decidual response compared to wildtype mice when treated with a high dose of progesterone and a low dose of estrogen followed by mechanical traumatization of the uterus ( Jeong et al. 2007 ). Proliferation of endometrial stromal cells is a key event before decidualization that is regulated by progesterone. SRC-2 drives this progesterone regulated proliferation by accelerating cellular glycolytic flux by upregulating the expression of the enzyme PFKFB3 ( Kommagani et al. 2013 ). This increase in glycolytic flux is necessary to provide energy and needed building biomolecules for rapid cellular proliferation. Indeed, acute treatment of mice with a small molecule inhibitor of PFKFB3 has been shown to suppress the ability of the animals to mount an endometrial decidual response. Furthermore, microarray analysis from SRC-2 depleted uterine tissue after acute progesterone treatment revealed that SRC-2 is required for the induction of the progesterone transcriptional program. SRC-2 induced transcription factor 23 (TCF23) to modulate progesterone induced decidualization of endometrial stromal cells ( Kommagani et al. 2014 ). Total RNA-seq from endometrial stromal cells undergoing in vitro decidualization after knockdown of SRC-2 expression revealed a connection between retinoid transport and SRC-2 signaling. Integrating RNA-seq and ChIP-seq data identified Stimulated by Retinoic Acid 6 (STRA6) as an important target gene of progesterone and SRC-2 signaling ( Szwarc et al. 2018 ). Together, SRC-2 has been shown to function as an important coactivator that can regulate progesterone receptor-induced transcriptional programs during endometrial stromal cell decidualization. SRC-2 also regulates the differentiation of bone marrow stromal cells into adipocytes. The loss of SRC-2 expression in mice resulted in increased bone mass and increased trabecular bone volume. SRC-2 knockout mice also showed an increase in the number of osteoblasts and a decrease in the number of adipocytes within the bone marrow. Since SRC-2 functions as a transcriptional coactivator for PPARγ target genes, these effects can be attributed to a partial PPARγ resistance phenotype ( Mödder et al. 2009 ). Mesenchymal stromal cells isolated from the bone marrow can be differentiated into osteoblasts or adipocytes in vitro . Bone marrow stromal cells isolated from SRC-2 knockout mice and cultured in osteoblastic medium displayed increased osteoblast colony forming units compared to wildtype mice. This effect was further enhanced in the presence of the PPARγ ligand rosiglitazone with stromal cells from SRC-2 knockout mice resulting in three times more osteoblast colony forming units compared to wildtype cells. Loss of SRC-2 reduced adipogenesis in bone marrow stromal cells and reduced expression of PPARγ target genes such as adiponectin and aP2 (fatty acid binding protein 2). Chromatin immunoprecipitation analysis found an increase in SRC-2 occupancy at the adiponectin PPRE (peroxisome proliferator response element) following rosiglitazone treatment in wildtype but not SRC-2 knockout bone marrow stromal cells. SRC-2 thus functions as a transcriptional coactivator mediating PPARγ effects on adipogenic genes in bone marrow stromal cells. SRC-2 also has been shown to be an important mediator of paracrine signaling between cardiomyocytes and endothelial cells during pressure overload injury in the heart ( Suh et al. 2017 ). In vitro , human umbilical vein endothelial cells (HUVECs) treated with conditioned media from cardiomyocyte-specific SRC-2 knockout cells displayed reduced proliferation and tube formation ability and this inhibition was rescued by treatment with exogenous vascular endothelial growth factor (VEGF). Furthermore, SRC-2 coactivated GATA-binding protein 4 (GATA-4) and hypoxia inducible factor (HIF)-1α and −2α in the cardiomyocytes to regulate VEGF expression and secretion. In this way SRC-2 mediated regulation of VEGF expression in cardiomyocytes stimulates endothelial cell proliferation and tube formation. SRC-3 is highly expressed in endothelial cells and vascular smooth muscle cells. Similar to SRC-1, SRC-3 is also required for estrogen induced vascular repair after injury. SRC-3 knockout mice displayed a deficiency in neointimal inhibition and increased cell proliferation after estrogen treatment in ovariectomized mice after carotid artery ligation ( Yuan et al. 2002 ). In vitro studies have shown that decreased SRC-3 expression in HUVECs reduced tube formation on matrigel and blocked their barrier function ( Al-Otaiby et al. 2012 ). SRC-3 also is important for endothelial cell migration and proliferation. Further explanation for the molecular action of SRC-3 mediated regulation of endothelial cell migration and tube formation came from a study by Wang et al. They found that extracellular signal-regulated kinase 3 (ERK3) signaling in endothelial cells upregulated the interaction of SRC-3 and CREB-binding protein (CBP) with the transcription factor specificity protein 1 (Sp1), leading to the upregulation of vascular endothelial growth factor receptor 2 (VEGFR2) expression ( Wang et al. 2014 ). This increased expression of VEGFR2 in endothelial cells promoted HUVEC proliferation, migration, and tube formation. In addition to endothelial cell migration, SRC-3 also regulated the migration of fibroblasts ( Al-Otaiby et al. 2012 ). SRC-3 function also has been shown to be important for tissue repair. Complete healing of skin wounds in SRC-3 knockout mice is impaired compared to wildtype controls ( Al-Otaiby et al. 2012 ). Histological analysis of these mice showed reduced neoangiogenesis and decreased infiltration of immune cells, namely macrophages and monocytes, in skin wounds. SRC-3 also regulates paracrine signaling between endothelial cells and tissue epithelial cells through modulation of fibroblast growth factor (FGF) signaling. FGF10 ligand produced by stromal cells that acts on epithelial cell FGFR2b is known to be important in wound re-epithelialization and angiogenesis ( Zhang et al. 2006 ; Gurtner et al. 2008 ). Expression of FGF10 and FGFR2b were both reduced in skin wounds in SRC-3 heterozygous mice, again highlighting the role of SRC-3 in regulating paracrine signaling within tissue stroma. SRC-3 regulates inflammatory signals within immune cells via suppression of cytokine release/production. Consequently, SRC-3 knockout mice are susceptible to endotoxic shock after LPS treatment as a result of increased production of proinflammatory cytokines including tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6) ( Yu et al. 2007 ). While mRNA levels of TNFα and IL-1β were unchanged in peritoneal macrophages between wildtype and SRC-3 knockout mice after LPS treatment, protein levels of TNFα and IL-1β were increased in SRC-3 knockout mice. This led to the discovery of a new role for SRC-3 as a ‘translational repressor’ of inflammation. SRC-3 brings about translation repression by interacting with the RNA binding proteins, T cell intracellular Ag-1 (TIA-1) and T cell intracellular Ag-1 related (TIAR), promoting their association with the AU rich regions within the TNFα mRNA. SRC-3 been shown to regulate phagocytosis in macrophages and neutrophils. In E. coli induced peritonitis, SRC-3 expression in peritoneal macrophages enhanced bacterial clearance ( Chen et al. 2010 ). Through its transcriptional coactivation function, SRC-3 upregulated the expression of the antioxidant enzyme, catalase and the macrophage surface receptor, SR-A which led to increased bacterial phagocytosis and decreased macrophage apoptosis. Collectively, SRC-3 function is important for endothelial cell angiogenesis and wound healing, fibroblast migration, and inflammatory signaling in immune cells.

Paracrine signaling between stromal and epithelial cells by cytokines, growth factors and hormones have been shown to regulate the crosstalk between tissue stroma and parenchyma. Previous studies have shown that steroid hormones such as estrogens, progestins and androgens play key roles in regulating stromal-epithelial interactions during tissue development and healing. Estrogen signaling is important for breast and prostate development ( Mueller et al. 2002 ) and wound healing ( Horng et al. 2017 ) and has beneficial effects on tissue vasculature ( White 2002 ), while progesterone hormone signaling is required for endometrial cell decidualization ( Dunn et al. 2003 ). One of the most well characterized examples of tissue paracrine signaling is during the process of endometrial decidualization and embryo implantation. The decidualization reaction is essential for successful implantation of the embryo. After embryo implantation, the decidua further develops to aid placentation. Paracrine signaling between the uterine stroma and epithelium plays an important role in making the uterus receptive for implantation. Studies using uterine tissue recombination and conditional deletions of estrogen and progesterone receptors have found that stromal ER signaling is required for uterine epithelial cell proliferation ( Pawar et al. 2014 ). One proposed mechanism by which this occurs is through paracrine signaling via stromal ER induced expression of insulin growth factor (IGF) that signals through IGF-1 receptors on the epithelium. Indian hedgehog (IHH), leukemia inhibitory factor (LIF) and heparin-binding epidermal growth factor (Hb-EGF) are other paracrine effectors that regulate uterine receptivity for embryo implantation. Epithelial PR regulates IHH production which acts by regulating stromal COUP-TFII signaling to promote decidualization. In another example, conditional deletion of the ER gene in the uterine epithelial compartment leads to loss of LIF production. Furthermore, the transcription factor signal transducer and activator of transcription 3 (STAT3) acts downstream of LIF in the epithelium to induce stromal cell proliferation by secretion of Hb-EGF. Mice lacking uterine epithelial specific STAT3 show decreased phosphorylation of the EGF receptor in the uterine stroma after implantation. Importantly, exogenous administration of EGF family effectors rescues the stromal cell proliferation defect in the mice lacking STAT3 in the uterine epithelium. Together, these results highlight the importance of steroid hormone signaling in regulating paracrine signaling between the tissue stroma and epithelial cellular compartments.

The steroid receptor coactivators (SRCs) consist of a family of three proteins. They function as transcriptional coactivator platform proteins that form a base for the assembly of multi-subunit protein complexes on transcription factors (TFs) at promoters to stimulate transcription ( Xu et al. 2009 ). The three family members SRC-1, SRC-2 and SRC-3 share about 60% sequence homology with each other. They contain three main functional domains consisting of the basic helix-loop-helix (bHLH)/Per/Arnt/Sim (bHLH-PAS) domain, the nuclear receptor interaction domain (RID) that contains two LXXL motifs (where X is any amino acid) and, two C-terminal activation domains termed AD1 and AD2. While there is some functional redundancy among the three family members, they also have important and distinct roles. They are the main coactivators required for the action of nuclear hormone receptors such as estrogen (ER), androgen (AR), progesterone (PR), and glucocorticoid receptors (GR). They also serve as coactivators for other nuclear receptors such as the retinoic acid receptor (RAR), the peroxisome proliferator-activated receptor-γ (PPARγ) as well as other transcription factors including NF-κB. While whole body knockout of any of the three SRCs do not result in embryonic lethality, studies have shown clear defects within the reproductive and hormone responsive tissues in the knockout animals ( Dasgupta et al. 2014 ). SRC-1 knockout mice show a significant decrease in uterine, prostate, testes and mammary tissue growth in response to hormone stimulation with no overt defects in viability and fertility. SRC-1 is also the major coactivator for ER and PR within the uterus and SRC-1 knockout leads to a 60% decrease in uterine growth. In contrast, SRC-2 knockout mice are hypofertile as a result of substantial reproductive tissue defects. Male SRC-2 mice show defective spermatogenesis and testicular degeneration while female SRC-2 knockout mice have major defects in uterine decidual response in addition to placental hypoplasia and strongly impaired embryo implantation. SRC-2 is also important for progesterone-induced mammary ductal branching and alveologenesis. SRC-3 knockout mice display abnormal reproductive physiology, postnatal growth retardation, and delayed puberty. SRC-3 functions as the major coactivator for ER and PR in mammary luminal epithelial cells and loss of SRC-3 leads to reduced mammary ductal branching and alveologenesis. Collectively, all three SRCs are important for tissue development. Global knockouts of the SRC family members in mice have highlighted the importance of the steroid receptor coactivators in regulating energy metabolism ( Dasgupta et al. 2014 ). Whole body knockout of SRC-1 in mice causes an increased risk of obesity due to decreased energy expenditure. SRC-1 null mice also display hypoglycemia due to impaired hepatic gluconeogenesis and SRC-1 directly regulates gene expression of key rate limiting enzymes such as pyruvate carboxylase, phosphoenolpyruvate carboxykinase and fructose-1, 6-bisphosphatase involved in gluconeogenesis. Conversely, SRC-2 null mice are protected from high fat diet induced obesity and demonstrate increased insulin sensitivity, higher lipolysis, and decreased fat absorption. SRC-2 null mice also exhibit phenotypes similar to Von Gierke’s disease due to reduced hepatic glucose release as a result of decreased expression of the glucose 6 phosphatase enzyme. Additionally, SRC-2 also plays an important role in coordinating whole-body energy homeostasis. It does this by stimulating fatty acid absorption from the gut through coactivation of farnesoid X receptor (FXR) to increase expression of bile salt export pump (BSEP). In contrast, knockout of SRC-3 in mice resembles human CACT metabolic myopathy ( York et al. 2012 ). SRC-3 in skeletal muscle regulates expression of the long-chain fatty acid transporter, carnitine/acylcarnitine translocase (CACT) and loss of SRC-3 leads to accumulation of long chain acyl carnitines in skeletal muscle. Thus, all three SRCs regulate different aspects of energy metabolism. All three SRCs also have been shown to promote cancer and metastasis in hormone dependent cancers ( Rohira & Lonard 2017 ). SRC-1 expression correlates with Her2 and c-myc expression and reduced disease-free survival in ER negative breast cancer. SRC-1 also promotes breast cancer cell invasion and migration by regulating expression of genes important for epithelial to mesenchymal transition (EMT) and is required for breast cancer metastasis. Indeed, knockout of SRC-1 in a PyMT mouse model for breast cancer did not affect tumor initiation and growth but significantly reduced metastasis to the lung. SRC-1 also is overexpressed in endocrine and aromatase inhibitor resistant breast tumors and plays a role in the development of chemo-resistance. In addition to breast cancer, SRC-1 mRNA expression is significantly upregulated in endometrial cancer. SRC-1 expression also is elevated in androgen-dependent prostate cancer and is associated with increased tumor aggressiveness. SRC-2 is amplified in prostate cancer and increased expression is associated with higher metastasis and recurrence in prostate cancer patients. SRC-2 loss causes inhibition of prostate cancer cell growth and metastasis and SRC-2 promotes prostate cancer metastasis by markedly regulating cancer cell metabolism. SRC-2 mRNA level is also increased in endometrial cancer. Conversely, SRC-2 functions as a tumor suppressor for chemical carcinogen induced liver cancer. SRC-3 is overexpressed and/or amplified in breast cancer and is associated with decreased disease-free survival. SRC-3 is important for breast tumor growth and metastasis. Overexpression of SRC-3 in a MMTV mouse model induces spontaneous tumor formation in the mammary gland. Additionally, SRC-3 increases lung, liver, prostate and ovarian tumor growth. SRC-3 also increases lung cancer metastasis and ovarian cancer cell motility and migration. With their important roles in promoting tumor cell growth and metastasis the SRCs serve as a new class of targets for therapeutic development. Indeed, small molecule compounds (SI-2/SI-12) that represses SRC transcriptional activity are capable of reducing breast ( Song et al. 2016 ), thyroid ( Lee et al. 2020 ) and pancreatic ( Song et al. 2019 ) tumor growth.

Tissue stroma carries out essential functions in tissue development and tissue repair after injury. Research in the past few years has highlighted the pathological role of the stroma in maladaptive wound healing, chronic inflammation, tissue fibrosis and in diseases such as cancer. The tumor stroma has been shown to promote tumor cell proliferation, migration, and metastasis. Cancer associated fibroblasts promote tumor growth while tumor associated macrophages create an immunosuppressive microenvironment allowing the tumor to proliferate and resist therapy ( Arendt et al. 2010 ). Efforts are currently underway to understand the crosstalk between the tumor stroma and cancer cells within the tumor microenvironment in order to identify and design more effective therapies. Conversely, the tissue stroma also is important for normal tissue healing as evidenced by the stromal cells in the skin that are essential mediators of the wound healing response. Efforts are also currently underway to determine the mechanisms underlying tissue fibrosis in order to develop treatments for fibrotic diseases such as lung and kidney fibrosis. The SRCs are ubiquitously expressed in stromal cells and have been shown to have important regulatory roles in mediating stromal cell functions ( Fig. 1 ). More recent work in our lab has shown that an SRC stimulator functions through cardiac fibroblasts and immune cells to protect cardiac function after a myocardial infarction ( Mullany et al. 2020 ). In conclusion, this review highlights the importance of further examining the role of the SRCs in stromal responses in normal and pathologic physiology, tissue healing, and oncogenesis to help develop new therapeutic strategies.