The Krüppel-like factors in female reproductive system pathologies.

Since KLF expression is ubiquitous yet known reproductive system pathologies appear to involve select subsets of KLFs ( Table 1 ), functional redundancies and compensatory regulation among KLFs must exist to ensure robust physiological responses to cellular perturbations for maintaining homeostasis. Recent elegant studies have demonstrated this concept for KLF3 and KLF8 in a non-reproductive (i.e., erythroid) system ( Eaton et al ., 2008 ; Funnell et al ., 2013 ). The lack of distinct uterine phenotypes in mouse knockout models for several KLF genes support this concept in the reproductive tract. A prime example involves the highly-related members KLF9 and KLF13. Albeit a definitive conclusion is limited by the lack of functional studies in mice deficient in both KLFs, support for a KLF9/KLF13 genetic interaction comes from findings that Klf9 -null mouse uteri at peri-implantation displayed increased Klf13 expression, which was confirmed in siKLF9 -targeted human endometrial stromal cells ( Pabona et al ., 2010 ). Moreover, Klf13 -null mice are reproductively normal, perhaps due to the accompanying increase in nuclear KLF9 protein levels shown for Klf13 -null endometrial cells (Heard et al ., 2013). Thus, the absence of an association between KLF13 and any reproductive dysfunctions reported to date ( Table 1 ) may be a consequence of the placement of KLF9 at a higher functional hierarchy relative to KLF13. In this scenario, potential transcriptional dysregulation that may occur with KLF13 loss-of-expression is abrogated by the compensatory actions of KLF9. The co-reduction of KLF9 and KLF4 expression noted in endometrial cancer and in endometriosis and those of KLF9 and KLF11 in endometriosis and in leiomyoma ( Table 1 ) on the other hand, support the concept of distinct programs of gene expression being controlled by these KLFs. Alternatively, this may indicate that there is an obligatory pathway that is mediated by both KLFs occurring through a linear mechanism. There is evidence for the latter possibility, at least for KLF9 and KLF4. KLF9 siRNA knockdown in the human endometrial carcinoma Ishikawa cell line reduced KLF4 transcript levels ( Simmons et al., 2011 ) and conversely, KLF9 over-expression in HEC-1A cells induced KLF4 gene expression ( Simmen et al ., 2008 ); these observations are in accord (albeit yet to be proven) that KLF4 serves as a downstream target of KLF9 either directly or indirectly. Parallel transcriptome and ChiP-Seq analyses of uterine cells subjected to siKLF9 and siKLF4 targeting, alone and in combination, will be required to identify unique and shared networks regulated by both KLFs and could provide insight into whether KLF4 is an early target of KLF9. Importantly, such studies may allow the identification of an obligate response (gene target, signaling pathway) mediated by both. In regards to KLF9 and KLF11, there are limited data to support or refute redundant functions; however, they are likely to differentially mediate PGR-driven transcriptional events in uterine cells based on their distinct reproductive phenotypes upon targeted gene inactivation ( Klf11 -null mice breed normally and are fertile in contrast to Klf9 -null mice which are subfertile) (Song et al. , 2010; Simmen et al ., 2004 ) and the distinct mechanisms by which they mediate PGR transactivity ( Zhang et al ., 2003 ; Yin et al ., 2010 ). The opposing actions of KLF4 and KLF15 in uterine epithelial cells constitute additional support for non-redundant functions of KLF family members. In these cells, KLFs 4 and 15 are inversely expressed, and are found to discretely regulate initiation of DNA synthesis by virtue of their distinct responses to E- and P-treatments ( Ray & Pollard, 2012 ). By inhibiting E-enhanced transcription of the DNA synthesis initiator protein minichromosome maintenance-2, KLF15 functions as a downstream mediator of P-inhibition of the cell cycle. What factors direct the inverse expression of KLFs 4 and 15 and their opposing responses to steroid hormones in the uterine epithelium have yet to be determined. Clearly, the biology underlying optimal uterine function involving KLF regulatory networks is wide-open for further investigations.

In the last decade, multiple molecular pathways mediated by KLFs have been elucidated in uterine and ovarian cells and tissues. Nevertheless, direct evidence linking described KLF effects to health outcomes and disease states remain elusive. How may we address this gap in knowledge? In most cases, the difficulty lies in the absence of mouse models that recapitulate the human disease and in the possible biological redundancies among subsets of KLFs that may prevent abnormal responses to be gleaned when one KLF is absent. Thus, it is imperative to establish which subsets of KLFs compensate for each other, using relevant cell lines in vitro by siRNA targeting and by characterizing uterine (or ovarian)-targeted KLF-combination knockouts in vivo . Many of the mouse mutants for KLFs have modest or no reproductive phenotypes when they survive through adulthood (e.g. Klf9 , Klf11 , and Klf13 null mice). For other KLFs, homozygous disruptions result in early embryo (for Klf4, Klf5 and Klf6 ), in utero (for Klf2 ) and neonatal (for Klf7 ) lethality ( Wani et al ., 1999 ; Matsumoto et al ., 2006 ; Laub et al ., 2006 ; Ema et al ., 2008 ). For these KLFs, therefore, conditional mutations using uterine epithelial, stromal and myometrial-specific promoters driving the Cre-recombinase may serve as a powerful strategy for studying gene function in each cell type. Such studies are anticipated to be labor-intensive and complex, given that the uterus has multiple cellular compartments and several KLFs exhibit preferential cellular expression (e.g., KLF9 in endometrial stroma and myometrium) ( Simmen et al ., 2004 ). Indeed, the complexity of ‘teasing out’ the details of KLF signaling in each compartment is best illustrated when one considers that for the P/PGR signaling pathway alone, distinct KLFs are involved either as regulators or integrators of P/PGR transactivity, albeit not necessarily under the same physiological contexts ( Fig. 4 ). To date, the proliferative, survival, and pro-/anti-inflammatory molecular signatures elicited by each KLF family member when null-mutated in specific uterine compartments have not been defined. The power of increasingly sophisticated approaches such as ChiP-Seq, various ‘omics’ technologies and precise genome editing methodologies using engineered nucleases offered by the clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) proteins should be harnessed to address this question. So why study KLFs in the face of their seeming complexity? The data presented in this review documenting: 1) their association with many reproductive disorders, whose etiologies remain not well-understood; 2) their control of a plethora of signaling pathways; and 3) the considerable diversity of their target genes due to their ability to act as transcriptional activators or repressors, collectively suggest their prominent roles as integrators of uterine (and ovarian) biology. Perhaps an exciting direction for KLF research is one that focuses on their transcriptional roles in uterine and ovarian stem cell biology. It is well-known that the endometrium displays dramatic regenerative properties, estimated to occur ~400-times during a woman’s reproductive years; these have been linked to the presence of adult stem cells displaying key properties of mesenchymal stem cells ( Figueira et al., 2011 ; Spitzer et al., 2012 ). In a recent study, Taylor and colleagues ( Sakr et al ., 2014 ) demonstrated that mesenchymal stem cells are recruited to endometriosis lesions and that reduction of this recruitment can diminish lesion incidence. Similarly, a small population of cells (~1% of tumor cells) showing stem-progenitor properties was found to be essential for E+P-dependent growth of uterine leiomyomas ( Ono et al., 2012 ). Interestingly, the growth of this cell population involves ESR/PGR and Wnt signaling pathway cross-talk via E+P-induced β-catenin translocation, leading to Axin2 promoter activation ( Ono et al., 2013 ). Since loss of KLF11 expression is associated with increased PGR signaling and proliferation of leiomyoma cells ( Yin et al., 2010 ), it is tempting to consider that inhibition of the aberrant expansion of myometrial smooth muscle stem cells by KLF11 may avert tumor initiation and leiomyoma. How will understanding the biology of KLFs lead to novel and more effective therapies for female reproductive disorders? To date, treatment options for most uterine disorders involve aromatase inhibitors and progestins; however, prolonged treatments with these agents can result in drug resistance, with disease recurring often times after cessation of treatment. If current data indicating that KLFs integrate P/PGR and E/ESR cross-talk with Notch and Wnt pathways to control aberrant stem/progenitor cell proliferation, are verified, it may be possible to develop non-steroidal treatments that target specific ‘stemness’ factors such as the Notch ligand Jagged1, that promote the survival of this subpopulation and hence, progression/recurrence of uterine pathologies. Thus, targeting Notch signaling with γ-secretase inhibitors that inhibit the intracellular localization of transcriptional mediator Notch intracellular domain may offer a viable therapeutic strategy. A proof-of-concept for the latter has been recently demonstrated for uterine serous carcinoma in a human xenograft model in mice ( Groeneweg et al ., 2014 ). In a recent report, small molecule inhibitors of the expression of the colorectal cancer oncogene KLF5 were identified by high-throughput screening of compound libraries ( Bialkowska et al., 2011 ). The isolated compounds, screened using a rat intestinal cell line stably expressing a luciferase reporter driven by the human KLF5 promoter, reduced endogenous KLF5 protein levels and decreased the viability of a number of colorectal cancer cell lines. A similar strategy may also be employed to elude reproductive pathologies, although compounds promoting, rather than inhibiting, KLF expression will need to be identified since uterine pathologies are mostly associated with reduced KLF expression ( Table 1 ). Such approaches could yield novel research outcomes valuable for translation into the clinic. Finally, it is worth noting that the major male reproductive disease namely prostate cancer is also highly associated with dysfunctions in numerous KLFs including KLF4 ( Wang et al., 2010 ), KLF5 ( Frigo et al., 2009 ), KLF6 ( Narla et al., 2001 ), KLF8 ( He et al., 2013 ) and KLF9 ( Shen et al., 2014 ). Importantly, a number of signaling pathways reported for KLF (dys) regulation of prostate epithelial cell proliferation, differentiation and survival overlap with those elucidated for KLF-mediated uterine function. In particular, KLFs have been reported to participate in androgen receptor-dependent signaling ( Liu et al ., 2012 ; He et al ., 2013 ), the male counterpart of PGR/ESR signaling in females; in regulating Hedgehog pathway components ( Leow et al ., 2009 ); and in stem cell signaling involving the Notch pathway ( Oklem et al ., 2014 ). However, no KLFs have been demonstrated so far to be indispensable for spermatogenesis.

It is notable that for those mice with global null-mutations of specific KLFs (e.g. KLF9, KLF11, KLF13) and surviving through adulthood, an ovarian phenotype characterized by dysfunctions in steroid hormone synthesis is not manifested throughout the reproductive years ( Simmen et al ., 2004 ; Zeng et al ., 2007; Heard et al ., 2012 ; Daftary et al ., 2013 ). This finding is not congruent with the demonstrated regulation of several key steroidogenic genes transcript levels (LDLR, StAR and CYP11A) by KLF13 in ovarian granulosa cells ( Natesampillai et al. , 2008 ). Interestingly, the pathologic ovary (i.e., ovarian carcinoma) is characterized by reduced (KLF2, KLF4, KLF6) and enhanced (KLF5, KLF8) expression of several KLFs; contradicting results have been reported for KLF9 ( Fig. 2B ). Analyses of currently identified target genes associated with dysregulation of distinct KLF expression in ovarian cancer cells revealed perturbations in those related to proliferation and differentiation (cyclin D1); apoptosis (Bcl2, Bax, survivin); epithelial-mesenchymal interactions (E-cadherin, vimentin, Extracellular matrix receptor); stem cell differentiation (USP44, ErbB), and angiogenesis (VEGF). These findings raise important questions on how KLFs alone or together may integrate the physiological processes in the ovary and whether pathways defined for uterine pathologies in which multiple KLFs (e.g., KLF4, KLF5) are similarly dysregulated, may be relevant to ovarian diseases.

The human uterus has a unique role in the successful transmission of germ line DNA to guarantee the propagation of the human species. Biologically, it is destined to provide the fertilized egg with a ‘nurturing’ environment for its development and maturation into a complex entity with unique capabilities to eventually function on its own. Defects in the proper development and function of the uterus present a major hurdle to reproduction. Moreover, various uterine-related pathologies including endometrial and cervical carcinoma, endometriosis, and leiomyoma may arise post-puberty to further contribute to infertility. The steroid hormones estrogen (E) and progesterone (P), working through their cognate nuclear receptors [estrogen receptor (ESR) 1 and ESR2; progesterone receptor (PGR) -A and PGR-B isoforms] are major regulators of uterine development and function ( Hamilton et al ., 2014 , Kim et al ., 2013 ). Their multi-faceted transcriptional pathways involve interactions with numerous nuclear co-regulators ( Sangupta & O’Malley, 2014 ) and result in altered levels of signaling molecules that act through paracrine and autocrine circuits. The underlying mechanism(s) for the autonomous and collective behavior of the multiple cell types of the uterus to maintain function, however, continues to be a work-in-progress, given recent discoveries of new participants and targets. In this review, we highlight emerging evidence documenting the participation of the multi-member Krüppel-like factor (KLF) family of transcription factors and the dynamics of their transcriptional networks and roles in cellular communication in select uterine pathologies. The association of KLFs in ovarian carcinoma is similarly presented since the ovary is the major source of the nuclear receptor ligands E and P and because ovarian-related infertility is a major problem in reproductive medicine. Disentangling the various mechanistic points of action of KLFs in these pathologies may aid in the identification of key parameters for optimal reproductive function and contribute to the development of novel treatment strategies and clinical applications to address reproductive disorders.

Factors that contribute to the aberrant expression and activity of KLFs in the reproductive tract leading to pathology have not been well-characterized, in contrast to other systems. In embryonic stem cells, induction of KLF2 by Oct4 and of KLF4 by LIF has been demonstrated, reinforcing these KLFs’ function in stem cell renewal ( Hall et al., 2009 ). KLF4 expression was suppressed by transcription factor FOXO in B-lymphocytes ( Yusuf et al ., 2008 ) and by an inhibitor of notch signaling in the mouse gastrointestinal tract ( Zheng et al., 2009 ), and conversely, was induced by Notch 1 intracellular domain in ocular surface epithelia ( Zhang et al., 2013 ). KLF6 expression was stimulated by IGF-1 in human colon cancer cell lines ( Bentov et al ., 2008 ) and the binding of carbohydrate response element-binding protein (ChREBP), a glucose-activated transcription factor, induced KLF10 promoter activity and expression in rat hepatocytes ( Iizuka et al., 2011 ). The identity of factors that regulate KLF expression in uterine cells is currently limited to that for KLF9 in human endometrial stromal cells; in these cells, BMP2 inhibited KLF9 expression indirectly through KLF13 ( Pabona et al., 2010 ) while E and P had no influence on its expression ( Pabona et al., 2012 ). In ovarian granulosa cells, IGF1 and LH were reported to increase KLF13 expression ( Natesampillai et al. , 2008 ). The comprehensive analyses of cellular components responsible for maintaining KLF expression will be required to understand and ultimately manipulate KLF regulatory circuits for optimal reproductive function.

The growing evidence for the functional and correlative association of KLFs in various female (and male) reproductive pathologies underscores the importance of extending and expanding current knowledge of this multi-faceted transcription factor family in reproductive health. New possibilities for targeting KLFs may soon be available from reproductive systemwide analyses of KLF signaling. Other reproductive pathologies including preeclampsia, fallopian tube cancers, and recurrent pregnancy loss as well as male infertility may similarly benefit from an understanding of KLF biology.

The Specificity Protein (SP)-related Krüppel-like factors (KLFs) is a 17-member family of DNA-binding transcriptional regulators of cellular proliferation, survival, differentiation, pluripotency and epithelial-mesenchymal interactions ( Suske et al ., 2005 ). We refer the reader to recent excellent reviews on this family ( Tetrault et al ., 2013 ; Knoedler & Denver, 2014 ; Limame et al. , 2014 ), which now also includes multiple biologically active KLF splice isoforms ( Camacho-Venegas et al ., 2013 ) and the related gene KLF18 that is present in the sequenced genomes of most placental mammals ( Pei & Grishin, 2013 ). KLF proteins are characterized by a conserved DNA-binding domain with three tandem C 2 H 2 -type zinc finger motifs at the carboxy-terminus and which recognizes the GT/GC box or CACCC element sites in promoter/5′ regulatory and enhancer regions ( Fig. 1A ). In contrast to the carboxy-termini, the amino-terminal regions of member proteins are highly variable in length and sequence and contain domains (including acidic transactivation domains, Sin-3 interacting repressor domains and CtBP2 interacting repressor domains) that interact with specific co-activators and co-repressors ( Kaczynski et al ., 2003 ); the diversity in this region is thought to confer unique functions to each family member. Figure 1B illustrates the sequence homologies between the two highly-related family members KLF9 and KLF13, where their respective C-terminal domains display highest similarities for both mouse and human proteins. Based on their phylogenetic relationships ( Limame et al ., 2014 ), KLF members can be categorized into three sub-groups ( Fig. 1C ). Interestingly, proteins within the same categories do not typically exhibit similar functions and tissue expression (discussed below), reflecting their distinct regulation, transcriptional activator or repressor roles, and the likely diversity of their interacting proteins under tissue-specific contexts.