Endocytosis-dependent lysosomal degradation of Src induced by protease-activated receptor 1.

Src plays a critical role in regulating cellular responses induced by protease-activated receptor 1 (PAR1). Here, we found that PAR1 activation induces lysosomal degradation of Src. Src is associated and trafficked together with activated PAR1 to early endosomes and then sorted to lysosomes for degradation. Blocking agonist-induced endocytosis of PAR1 by inhibition of dynamin activity suppresses PAR1-induced degradation of Src. However, Src activity is neither required for agonist-induced PAR1 internalization nor required for Src degradation upon PAR1 activation. We show that PAR1 activation triggers endocytosis-dependent lysosomal degradation of Src in both human embryonic kidney 293 and human umbilical vein endothelial cells. Our finding provides a new paradigm for how an irreversibly activated receptor regulates its downstream signalling.

RNA-activated DNA cleavage by the Type III-B CRISPR-Cas effector complex.

The CRISPR (clustered regularly interspaced short palindromic repeat) system is an RNA-guided immune system that protects prokaryotes from invading genetic elements. This system represents an inheritable and adaptable immune system that is mediated by multisubunit effector complexes. In the Type III-B system, the Cmr effector complex has been found to cleave ssRNA in vitro. However, in vivo, it has been implicated in transcription-dependent DNA targeting. We show here that the Cmr complex from Thermotoga maritima can cleave an ssRNA target that is complementary to the CRISPR RNA. We also show that binding of a complementary ssRNA target activates an ssDNA-specific nuclease activity in the histidine-aspartate (HD) domain of the Cmr2 subunit of the complex. These data suggest a mechanism for transcription-coupled DNA targeting by the Cmr complex and provide a unifying mechanism for all Type III systems.

Mouse forkhead L2 maintains repression of FSH-dependent genes in the granulosa cell.

The forkhead transcription factor forkhead box L2 (FOXL2) is expressed in granulosa cells of small and medium follicles in the mouse ovary. Foxl2 female knockout mice exhibit primordial follicle depletion and primary ovarian failure, but evidence from adult female conditional Foxl2 knockout mice suggests that FOXL2 may also play a significant role in maintenance of ovarian differentiation at stages beyond the primordial follicle and initial wave of folliculogenesis. We previously showed that human FOXL2 functions as a transcriptional repressor of several key genes involved in granulosa cell proliferation and differentiation, including steroidogenic acute regulatory protein (STAR), P450aromatase (CYP19A1 (CYP19)), P450scc (CYP11A1 (CYP11A)), and cyclin D2 (CCND2). To elucidate the role of mouse FOXL2, we determined its role in transcriptional regulation in Chinese hamster ovary (CHO) cells and then confirmed our findings in mouse granulosa cells. We found that mouse FOXL2 represses the activities of the mouse Star, Cyp19a1, Cyp11a1 promoters in CHO cells, but may not repress the Ccnd2 promoter, and identified the minimal mouse Star, Cyp19a1, and Cyp11a1 promoter regions responsive to FOXL2 regulation. We then knocked down Foxl2 in mouse granulosa cells using siRNA, which resulted in significantly increased expression levels of mouse Star, Cyp19a1, and Cyp11a1 but not Ccnd2. To increase Foxl2 expression levels, we generated a mouse Foxl2 lentiviral construct and used it to infect mouse granulosa cells. Following lentiviral infection, the expression levels of mouse Star, Cyp19a1, and Cyp11a1, but not Ccnd2, decreased significantly. These data confirm that mouse FOXL2 functions as a transcriptional repressor of key granulosa cell genes that influence ovarian development.

Adiponectin and its receptors modulate granulosa cell and cumulus cell functions, fertility, and early embryo development in the mouse and human.

OBJECTIVE: To study the expression and function of adiponectin and its receptors in mouse and human follicle cells and in early embryo development. DESIGN: Whole ovaries, granulosa cells, and cumulus-oocyte complexes isolated from immature mice before and during hormone-induced ovulation were used to analyze the expression of adiponectin, its receptors, and ovulation-related genes; human cumulus cells and granulosa cells were isolated from patients undergoing in vitro fertilization (IVF) procedures. SETTING: Multicenter. PATIENT(S): Women in IVF programs in Japan and the United States. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Expression of adiponectin receptors and fertility. RESULT(S): Adiponectin expression is absent/low in mouse and human granulosa cells and cumulus cells. Adiponectin receptors are hormonally regulated in mouse granulosa and cumulus cells in vivo and in culture. Adiponectin differentially alters the expression of Adipor1/Adipor2 as well as genes related to steroidogenesis, ovulation, and apoptosis in cumulus cells versus granulosa cells. Adiponectin enhances oocyte maturation and early embryo development in mouse and human IVF procedures. CONCLUSION(S): Adiponectin can modulate not only follicle growth but also embryo development in mice and humans.

Mutant Forkhead L2 (FOXL2) proteins associated with premature ovarian failure (POF) dimerize with wild-type FOXL2, leading to altered regulation of genes associated with granulosa cell differentiation.

Premature ovarian failure in the autosomal dominant disorder blepharophimosis-ptosis-epicanthus inversus is due to mutations in the gene encoding Forkhead L2 (FOXL2), producing putative truncated proteins. We previously demonstrated that FOXL2 is a transcriptional repressor of the steroidogenic acute regulatory (StAR), P450SCC (CYP11A), P450aromatase (CYP19), and cyclin D2 (CCND2) genes, markers of ovarian follicle proliferation and differentiation. Furthermore, we found that mutations of FOXL2 may regulate wild-type FOXL2, leading to loss of transcriptional repression of CYP19, similar to StAR. However, the regulatory mechanisms underlying these premature ovarian failure-associated mutations remain largely unknown. Therefore, we examined the effects of a FOXL2 mutant protein on the transcriptional repression of the CYP19 promoter by the full-length protein. We found that mutant FOXL2 exerts a dominant-negative effect on the repression of CYP19 by wild-type FOXL2. Both wild-type and mutant FOXL2 and can form homo- and heterodimers. We identified a minimal -57-bp human CYP19 promoter containing two potential FOXL2-binding regions and found that both wild-type and mutant FOXL2 can bind to either of these regions. Mutational analysis revealed that either site is sufficient for transcriptional repression by wild-type FOXL2, and the dominant-negative effect of mutant FOXL2, but these are eliminated when both sites are mutated. These findings confirm that mutant FOXL2 exerts a dominant-negative effect on wild-type FOXL2's activity as a transcriptional repressor of key genes in ovarian follicle differentiation and suggest that this is likely due to heterodimer formation and possibly also competition for DNA binding.

Relative expression of genes encoding SMAD signal transduction factors in human granulosa cells is correlated with oocyte quality.

PURPOSE: To determine the expression of SMAD transcripts in human granulosa cells. METHODS: Luteinized mural granulosa cells were harvested from forty women undergoing oocyte retrieval, and RNAs were isolated. SMAD expression levels were determined by polymerase chain reaction (PCR) and quantitative real-time PCR (q-RTPCR). RESULTS: SMAD1-7 and 9 are expressed in human granulosa cells, with SMAD2, 3 and 4 showing the highest expression levels. Peak estradiol (E2) levels correlated with the number of oocytes retrieved during IVF. Oocyte number showed no correlation with SMAD expression levels or ratios. Fertilization rates also did not correlate with the expression levels of individual SMADs, but did correlate with higher SMAD4:SMAD3 ratios (p = 0.0062) and trended with SMAD4:SMAD2 (p = 0.0698). CONCLUSIONS: SMAD transcripts are differently expressed in human granulosa cells, where they may mediate TGF-beta superfamily signaling during folliculogenesis and ovulation. Further, the relative expression ratios of SMAD2, 3 and 4 may differentially affect fertilization rate.

Minireview: roles of the forkhead transcription factor FOXL2 in granulosa cell biology and pathology.

The forkhead transcription factor (FOXL2) is an essential transcription factor in the ovary. It is important in ovarian development and a key factor in female sex determination. In addition, FOXL2 plays a significant role in the postnatal ovary and follicle maintenance. The diverse transcriptional activities of FOXL2 are likely attributable to posttranslational modifications and binding to other key proteins involved in granulosa cell function. Mutations of FOXL2 lead to disorders of ovarian function ranging from premature follicle depletion and ovarian failure to unregulated granulosa cell proliferation leading to tumor formation. Thus, FOXL2 is a key regulator of granulosa cell function and a master transcription factor in these cells.

LATS1 phosphorylates forkhead L2 and regulates its transcriptional activity.

Forkhead L2 (FOXL2) is expressed in the ovary and acts as a transcriptional repressor of the steroidogenic acute regulatory (StAR) gene, a marker of granulosa cell differentiation. Human FOXL2 mutations that produce truncated proteins lacking the COOH terminus result in blepharophimosis/ptosis/epicanthus inversus (BPES) syndrome type I, which is associated with premature ovarian failure (POF). In this study, we investigated whether FOXL2's activity as a transcriptional repressor is regulated by phosphorylation. We found that FOXL2 is phosphorylated at a serine residue and, using yeast two-hybrid screening, identified LATS1 as a potential FOXL2-interacting protein. LATS1 is a serine/threonine kinase whose deletion in mice results in an ovarian phenotype similar to POF. Using coimmunoprecipitation and kinase assays, we confirmed that LATS1 binds to FOXL2 and demonstrated that LATS1 phosphorylates FOXL2 at a serine residue. Moreover, we found that FOXL2 and LATS1 are coexpressed in developing mouse gonads and in granulosa cells of small and medium follicles in the mouse ovary. Last, we demonstrated that coexpression with LATS1 enhances FOXL2's activity as a repressor of the StAR promoter, and this results from the kinase activity of LATS1. These results provide novel evidence that FOXL2 is phosphorylated by LATS1 and that this phosphorylation enhances the transcriptional repression of the StAR gene, a marker of granulosa cell differentiation. These data support our hypothesis that phosphorylation of FOXL2 may be a control mechanism regulating the rate of granulosa cell differentiation and hence, follicle maturation, and its dysregulation may contribute to accelerated follicular development and POF in BPES type I.

Human forkhead L2 represses key genes in granulosa cell differentiation including aromatase, P450scc, and cyclin D2.

FOXL2 is expressed in granulosa cells (GC) of small and medium ovarian follicles, functions as a repressor of the human steroidogenic acute regulatory gene, a marker of a GC differentiation, and its mutation is associated with premature ovarian failure (POF) in women with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES), type I. We now report that FOXL2 also represses the transcription of aromatase, P450scc, and cyclin D2, three other key genes involved in GC proliferation, differentiation, and steroidogenesis, and that a FOXL2 mutation found in patients with BPES type I, also fails to repress aromatase transcription, further supporting a role for FOXL2 in follicle maturation.

Sumoylation of forkhead L2 by Ubc9 is required for its activity as a transcriptional repressor of the Steroidogenic Acute Regulatory gene.

Forkhead L2 (FOXL2) is a member of the forkhead/hepatocyte nuclear factor 3 (FKH/HNF3) gene family of transcription factors and acts as a transcriptional repressor of the Steroidogenic Acute Regulatory (StAR) gene, a marker of granulosa cell differentiation. FOXL2 may play a role in ovarian follicle maturation and prevent premature follicle depletion leading to premature ovarian failure. In this study, we found that FOXL2 interacts with Ubc9, an E2-conjugating enzyme that mediates sumoylation, a key mechanism in transcriptional regulation. FOXL2 and Ubc9 are co-expressed in granulosa cells of small and medium ovarian follicles. FOXL2 is sumoylated by Ubc9, and this Ubc9-mediated sumoylation is essential to the transcriptional activity of FOXL2 on the StAR promoter. As FOXL2 is endogenous to granulosa cells, we generated a stable cell line expressing FOXL2 and found that activity of the StAR promoter in this cell line is greatly decreased in the presence of Ubc9. The sumoylation site was identified at lysine 25 of FOXL2. Mutation of lysine 25 to arginine leads to loss of transcriptional repressor activity of FOXL2. Taken together, we propose that Ubc9-mediated sumoylation at lysine 25 of FOXL2 is required for transcriptional repression of the StAR gene and may be responsible for controlling the development of ovarian follicles.

Expression of forkhead transcription factors in human granulosa cells.

Members of the forkhead box O1 (FOXO) family of transcription factors are expressed in granulosa cells during various stages of follicle development, and evidence from rodent and other model systems suggests that they may be involved in regulating follicular activation and oocyte maturation. In this report, we show that FOXO1, FOXO3, and FOXO4 are expressed in human luteinized mural granulosa cells, which may suggest that these transcription factors are also involved in human folliculogenesis and luteinization.

Negative regulation of protease-activated receptor 1-induced Src kinase activity by the association of phosphocaveolin-1 with Csk.

Protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, has been correlated with cell proliferation. PAR1 is activated by the irreversibly proteolytic cleavage, internalized via clathrin-coated pits, and then sorted to lysosomes for degradation. Caveolae play important roles in both signaling transduction and internalization of several GPCRs. However, the role of caveolae in cellular signaling and trafficking of PAR1 is still unclear. In this study, we show that PAR1 was partially localized in caveolae. Disruption of caveolae by cholesterol depletion did not inhibit PAR1 internalization, indicating that internalization of PAR1 was not via caveolae. Of interest, activation of PAR1 resulted in the phosphorylation of caveolin-1, a principal component of caveolae, on tyrosine 14 by a Gi-linked Src kinase pathway and p38 mitogen-activated protein kinase. Analysis of immunoprecipitates from cells stimulated by PAR1 showed that phosphocaveolin-1 but not caveolin-1 with mutation at tyrosine 14 could bind to Csk. In addition, phosphocaveolin-1 could not bind to CskS109C mutant with the defective SH2 domain. These results indicated that phosphocaveolin-1 was associated with the SH2 domain of Csk in response to PAR1 activation. The association further resulted in a rapid decrease in Src kinase activity. Thus, PAR1-induced Src activation is negatively regulated by recruiting Csk through phosphocaveolin-1. Our results also reveal that phosphocaveolin-1 represents a novel effector of PAR1 to downregulate Src kinase activity. The downregulation of PAR1-induced Src activation mediated by phosphocaveolin-1 provides an additional mechanism for the termination of PAR1 signaling at its downstream molecules.

Opposing effects of beta-arrestin1 and beta-arrestin2 on activation and degradation of Src induced by protease-activated receptor 1.

Protease-activated receptor 1 (PAR1), a G protein-coupled receptor for thrombin, is irreversibly proteolytically activated. beta-Arrestin1 and beta-arrestin2 have been reported to have different effects on signal desensitization and transduction of PAR1. In this study, we investigated whether beta-arrestin1 and beta-arrestin2 regulate Src-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) induced by PAR1 in HEK 293 cells. Our results show that PAR1-mediated activation of Src and ERK1/2 in HEK 293 cells was increased with overexpression of beta-arrestin1 or depletion of beta-arrestin2. PAR1-mediated activation of Src and ERK1/2 in HEK 293 cells was decreased or eliminated with depletion of beta-arrestin1 or overexpression of beta-arrestin2. Furthermore, depletion of beta-arrestin2 blocked PAR1-induced degradation of Src. Thus, beta-arrestin1 and beta-arrestin2 have opposing roles in regulating the activation of Src induced by PAR1. beta-Arrestin2 also appears to promote PAR1-induced degradation of Src. This degradation of Src provides a possible mechanism for terminating PAR1 signaling.