gut-enriched Kruppel-like factor (Gklf) encodes a
member of the Kruppel family of transcription factors. Immunofluorescence revealed that Gklf is localized to the
nucleus and is found at highest levels in growth-arrested cells. This gene was found to be regulated by gonadotropins in DNA arrays.
NCBI Summary:
This gene encodes a protein that belongs to the Kruppel family of transcription factors. The encoded zinc finger protein is required for normal development of the barrier function of skin. The encoded protein is thought to control the G1-to-S transition of the cell cycle following DNA damage by mediating the tumor suppressor gene p53. Mice lacking this gene have a normal appearance but lose weight rapidly, and die shortly after birth due to fluid evaporation resulting from compromised epidermal barrier function. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Sep 2015]
Krüppel-like factor 4 plays a role in the luteal transition in steroidogenesis by down-regulating Cyp19A1 expression. Choi H et al. (2019) The transition from granulosa cell to luteal cell involves a change from estrogen production to predominantly progesterone production. We analyzed the role of Krüppel-like factor 4 (Klf4), a transcriptional repressor used to generate pluripotent cells, in that transition. After luteinizing hormone/human chorionic gonadotropin (LH/hCG) treatment of preovulatory follicles, a major but transient increase in Klf4 transcript levels was detected. Therefore we enquired whether Klf4 is involved in the rapid decline of aromatase, the key estrogen-producing enzyme, using preovulatory granulosa cells (GCs) obtained from PMSG-primed immature rat ovaries. Cyp19A1 expression in GCs transfected with FLAG- Klf4 or Klf4-specific siRNA was analyzed by real-time PCR and immunofluorescence staining. Cyp19A1 decreased when Klf4 was overexpressed, and Cyp19A1 and estradiol biosynthesis increased when Klf4 was knocked down. The mechanism by which Klf4 regulates Cyp19A1 expression was investigated using Cyp19A1 promoter-luciferase reporter assays and ChIP assays. The results revealed that the SF1-binding motif, but not the Sp1 binding element or the CACCC motif, was required for Klf4-mediated repression of Cyp19A1 promoter activity. Here, we showed that Klf4 suppressed endogenous Cyp19A1 transcript and protein production, and this resulted from direct binding of Klf4 to the SF1 recognition motif in the Cyp19A1 promoter. These findings suggest that Klf4 is a physiologic regulator of Cyp19A1 expression in response to the LH surge in preovulatory GCs, and that it has an essential role in the luteal transition in steroidogenesis.//////////////////
Krüppel-like factor 4 plays a role in the luteal transition in steroidogenesis by down-regulating Cyp19A1 expression. Choi H et al. (2019) The transition from granulosa cell to luteal cell involves a change from estrogen production to predominantly progesterone production. We analyzed the role of Krüppel-like factor 4 (Klf4), a transcriptional repressor used to generate pluripotent cells, in that transition. After luteinizing hormone/human chorionic gonadotropin (LH/hCG) treatment of preovulatory follicles, a major but transient increase in Klf4 transcript levels was detected. Therefore we enquired whether Klf4 is involved in the rapid decline of aromatase, the key estrogen-producing enzyme, using preovulatory granulosa cells (GCs) obtained from PMSG-primed immature rat ovaries. Cyp19A1 expression in GCs transfected with FLAG- Klf4 or Klf4-specific siRNA was analyzed by real-time PCR and immunofluorescence staining. Cyp19A1 decreased when Klf4 was overexpressed, and Cyp19A1 and estradiol biosynthesis increased when Klf4 was knocked down. The mechanism by which Klf4 regulates Cyp19A1 expression was investigated using Cyp19A1 promoter-luciferase reporter assays and ChIP assays. The results revealed that the SF1-binding motif, but not the Sp1 binding element or the CACCC motif, was required for Klf4-mediated repression of Cyp19A1 promoter activity. Here, we showed that Klf4 suppressed endogenous Cyp19A1 transcript and protein production, and this resulted from direct binding of Klf4 to the SF1 recognition motif in the Cyp19A1 promoter. These findings suggest that Klf4 is a physiologic regulator of Cyp19A1 expression in response to the LH surge in preovulatory GCs, and that it has an essential role in the luteal transition in steroidogenesis.//////////////////Role of Klf4 in the Regulation of Apoptosis and Cell Cycle in Rat Granulosa Cells during the Periovulatory Period. Choi H et al. (2018) In the ovary, the luteinizing hormone (LH) surge suppresses the proliferation and induces the luteinization of preovulatory granulosa cells (GCs), which is crucial for the survival of terminally-differentiated GCs. Krüppel-like factor 4 (Klf4) has been shown to play a role in regulating the cell cycle and apoptosis in various cell types. The rapid induction of Klf4 expressions by LH was observed in preovulatory GCs. To evaluate whether Klf4 affects GC proliferation and survival, primary rat GCs were isolated from pregnant mare serum gonadotropin-primed Sprague⁻Dawley rat ovaries and transfected with a Klf4 expression vector or Klf4-specific siRNA, followed by determination of the transcript levels of apoptosis-related and cell cycle-related genes. Cell proliferation, viability, and apoptosis were analyzed by BrdU incorporation, a Cell Counting Kit-8 assay, a bioluminescence caspase 3/7 assay, and flow cytometry. LH treatment increased Klf4 mRNA expression in preovulatory GCs. Transcripts of B-cell lymphoma 2 (Bcl-2) and cell cycle promoters (Cyclin D1 and Cyclin D2) decreased, whereas those of the cell cycle inhibitor, p21, increased. Altering the expression of Klf4 by overexpression or knockdown consistently affected the expression of Bcl-2 and Cyclin D1. In agreement with this, Klf4 overexpression reduced cell viability, increased the fraction of apoptotic cells, and arrested cell cycle progression in G1 phase. We conclude that Klf4 increases the susceptibility of preovulatory GCs to apoptosis by down-regulating Bcl-2, and promotes LH-induced cell cycle exit. It appears to be a key regulator induced by the LH surge that determines the fate of GCs in preovulatory follicles during the luteal transition.//////////////////MicroRNA-145 protects follicular granulosa cells against oxidative stress-induced apoptosis by targeting Krüppel-like factor 4. Xu L et al. (2017) Oxidative stress-induced follicular granulosa cell (GC) apoptosis plays an essential role in abnormal follicular atresia, which may trigger ovarian dysfunction. To investigate the role of microRNA (miR)-145 in the regulation of GC apoptosis and modulation of the apoptotic pathway in the setting of oxidative stress, we employed an H2O2-induced in vitro model and a 3-nitropropionic acid (NP)-induced in vivo model of ovarian oxidative stress. We demonstrated in vitro that miR-145 expression was significantly down-regulated in KGN cells and mouse granulosa cells (mGCs) treated with H2O2, whereas miR-145 over-expression attenuated H2O2-induced apoptosis in GCs. Moreover, miR-145 protected GCs against H2O2-induced apoptosis by targeting KLF4, which promoted H2O2-induced GC apoptosis via the BAX/BCL-2 pathway. Importantly, decreased miR-145 expression in the in vivo ovarian oxidative stress model promoted apoptosis by up-regulating KLF4 expression, whereas GC-specific miR-145 over-expression attenuated apoptosis by targeting KLF4. In conclusion, miR-145 protects GCs against oxidative stress-induced apoptosis by targeting KLF4.//////////////////
Expression regulated by
LH, Growth Factors/ cytokines
Comment
LH-induced Transcriptional Regulation of Klf4 Expression in Granulosa Cells Occurs via the cAMP/PKA Pathway and Requires a Putative Sp1 Binding Site. Choi H et al. (2020)Krüppel-like factor 4 (Klf4) plays an important role in the transition from proliferation to differentiation in a wide variety of cells. Previous studies demonstrated its critical role in the luteal transition of preovulatory granulosa cells (GCs). This study used cultured rat preovulatory GCs to investigate the mechanism by which luteinizing hormone (LH) regulates Klf4 gene expression. Klf4 mRNA and protein were rapidly and transiently induced by LH treatment, reaching peak levels after 45 min and declining to basal levels by 3 h. Pretreatment with the protein synthesis inhibitor cycloheximide had no effect on LH-stimulated Klf4 expression, indicating that Klf4 is an immediate early gene in response to LH. To investigate the signaling pathway involved in LH-induced Klf4 regulation, the protein kinase A (PKA) and protein kinase C (PKC) pathways were evaluated. A-kinase agonists, but not a C-kinase agonist, mimicked LH in inducing Klf4 transcription. In addition, specific inhibitors of A-kinase abolished the stimulatory effect of LH on Klf4 expression. Truncation of a Klf4 expression construct to -715 bp (pKlf4-715/luc) had no effect on transcriptional activity, whereas deletion to -402 bp (pKlf4-402/luc) dramatically reduced it. ChIP analysis revealed in vivo binding of endogenous Sp1 to the -715/-500 bp region and maximal transcriptional responsiveness to LH required the Sp1 binding element at -698/-688 bp, which is highly conserved in mice, rats, and humans. These findings demonstrate that Klf4 is activated by LH via the cAMP/PKA pathway and a putative Sp1 binding element at -698/-688 bp is indispensable for activation and suggest that Klf4 could be a target for strategies for treating luteal phase insufficiency induced by an aberrant response to the LH surge.//////////////////
Ovarian localization
Oocyte, Granulosa
Comment
Regulation of Kruppel-like factor 4, 9 and 13 genes and the steroidogenic genes LDLR, StAR and CYP11A in ovarian granulosa cells. Natesampillai S et al. Kruppel-like factors (KLF's) are important Sp1-like eukaryotic transcriptional proteins. The LDLR, StAR and CYP11A genes exhibit GC-rich Sp1-like sites, which have the potential to bind KLF's in multiprotein complexes. We now report that KLF4, KLF9 and KLF13 transcripts are expressed in and regulate ovarian cells. KLF4 and 13, but not KLF9, mRNA expression was induced and then repressed over time (P < 0.001). Combined LH and IGF-I stimulation increased KLF4 mRNA at 2 h (P < 0.01), whereas LH decreased KLF13 mRNA at 6 h (P < 0.05) and IGF-I reduced KLF13 at 24 h (P < 0.01) compared with untreated control. KLF9 was not regulated by either hormone. Transient transfection of KLF4, KLF9 and KLF13 suppressed LDLR/luc, StAR/luc and CYP11A/luc by 80 to 90% (P < 0.001). Histone-deacetylase (HDAC) inhibitors stimulated LDLR/luc by 5-6 fold and StAR/luc and CYP11A/luc activity by 2-fold (P < 0.001), and partially reversed suppression by all 3 KLF's (P < 0.001). Deletion of the zinc-finger domain of KLF13 abrogated repression of LDLR/luc. Lentiviral overexpression of the KLF13 gene suppressed LDLR mRNA (P < 0.001) and CYP11A mRNA (P = 0.003), but increased StAR mRNA (P = 0.007). Collectively, these data suggest that KLF's may recruit inhibitory complexes containing HDAC corepressors, thereby repressing LDLR and CYP11A transcription. Conversely, KLF13 may recruit unknown coactivators or stabilize StAR mRNA, thereby explaining enhancement of in situ StAR gene expression. These data introduce new potent gonadal transregulators of genes encoding proteins that mediate sterol uptake and steroid biosynthesis. Key words: KLF4, KLF9, KLF13, LDLR, StAR.
Follicle stages
Antral, Preovulatory
Comment
Liu HC, et al 2001 reported tha application of complementary DNA microarray (DNA chip) technology in the study of gene expression profiles during
folliculogenesis.
They used oligonucleotide microarray (DNA chip)-based hybridization
analysis to gain a comprehensive view of gene expression and regulation
involved in folliculogenesis.
Preantral follicles isolated from day 14 B6D2F-1 mice
were stimulated in vitro to form Graafian follicles. Total RNA extracted from
the mouse preantral and Graafian follicles were reverse transcribed, labeled
with digoxigenin-11-dUTP, and then hybridized with Clontech Atlas mouse cDNA
expression arrays for comparison. Of 588 known studied genes, 39 and 61 were detected in preantral follicles and in Graafian follicles, respectively, and 17 were highly
expressed consistently in both preantral and Graafian follicles. Performing
clustering analysis, 46 were upregulated as the follicles advanced to mature stages.
The KRUPPEL-LIKE FACTOR 4 is up-regulated in the Graafian follicles.