Aurélia E. Lewis

Molecular aspects of steroidogenic factor 1 (SF-1)

Steroidogenic factor 1 (SF-1, also called Ad4BP and NR5A1) is a nuclear receptor with critical roles in steroidogenic tissues, as well as in the brain and pituitary. In particular, SF-1 has emerged as an essential regulator of adrenal and gonadal functions and development. In the last few years, our knowledge on SF-1 has increased considerably at all levels, from the gene to the protein, and on its specific roles in different physiological processes. In this review, we discuss the current understanding on SF-1 with focus on the parameters that control the transcriptional capacity of SF-1 and the mechanisms that ensure proper stage- and tissue-specific expression of the gene encoding SF-1.

Identification of Nuclear Phosphatidylinositol 4,5-Bisphosphate-Interacting Proteins by Neomycin Extraction

Considerable insight into phosphoinositide-regulated cytoplasmic functions has been gained by identifying phosphoinositide-effector proteins. Phosphoinositide-regulated nuclear functions however are fewer and less clear. To address this, we established a proteomic method based on neomycin extraction of intact nuclei to enrich for nuclear phosphoinositide-effector proteins. We identified 168 proteins harboring phosphoinositide-binding domains. Although the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R-(Xn = 3–7)-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology or plant homeodomain modules. Proteins with no prior history of phosphoinositide interaction were identified, some of which have functional roles in RNA splicing and processing and chromatin assembly. The remaining proteins represent potentially other novel nuclear phosphoinositide-effector proteins and as such strengthen our appreciation of phosphoinositide-regulated nuclear functions. DNA topology was exemplar among these: Biochemical assays validated our proteomic data supporting a direct interaction between phosphatidylinositol 4,5-bisphosphate and DNA Topoisomerase IIα. In addition, a subset of neomycin extracted proteins were further validated as phosphatidyl 4,5-bisphosphate-interacting proteins by quantitative lipid pull downs. In summary, data sets such as this serve as a resource for a global view of phosphoinositide-regulated nuclear functions.

Deoxyribonucleic Acid Methylation Controls Cell Type-Specific Expression of Steroidogenic Factor 1

Steroidogenic factor 1 (SF1) is expressed in a time- and cell-specific manner in the endocrine system. In this study we present evidence to support that methylation of CpG sites located in the proximal promoter of the gene encoding SF1 contributes to the restricted expression pattern of this nuclear receptor. DNA methylation analyses revealed a nearly perfect correlation between the methylation status of the proximal promoter and protein expression, such that it was hypomethylated in cells that express SF1 but hypermethylated in nonexpressing cells. Moreover, in vitro methylation of this region completely repressed reporter gene activity in transfected steroidogenic cells. Bisulfite sequencing of DNA from embryonic tissue demonstrated that the proximal promoter was unmethylated in the developing testis and ovary, whereas it was hypermethylated in tissues that do not express SF1. Together these results indicate that the DNA methylation pattern is established early in the embryo and stably inherited thereafter throughout development to confine SF1 expression to the appropriate tissues. Chromatin immunoprecipitation analyses revealed that the transcriptional activator upstream stimulatory factor 2 and RNA polymerase II were specifically recruited to this DNA region in cells in which the proximal promoter is hypomethylated, providing functional support for the fact that lack of methylation corresponds to a transcriptionally active gene. In conclusion, we identified a region within the SF1/Sf1 gene that epigenetically directs cell-specific expression of SF1.

Ras Triggers Ataxia-telangiectasia-mutated and Rad-3-related Activation and Apoptosis through Sustained Mitogenic Signaling

Genetic evidence indicates that Ras plays a critical role in the initiation and progression of human thyroid tumors. Paradoxically, acute expression of activated Ras in normal rat thyroid cells induced deregulated cell cycle progression and apoptosis. We investigated whether cell cycle progression was required for Ras-stimulated apoptosis. Ras increased CDK-2 activity following its introduction into quiescent cells. Apoptotic cells exhibited a sustained increase in CDK-2 activity, accompanied by the loss of CDK-2-associated p27. Blockade of Ras-induced CDK-2 activity and S phase entry via overexpression of p27 inhibited apoptosis. Inactivation of the retinoblastoma protein in quiescent cells through expression of HPV-E7 stimulated cell cycle progression and apoptosis, indicating that deregulated cell cycle progression is sufficient to induce apoptosis. Ras failed to induce G1 phase growth arrest in normal rat thyroid cells. Rather, Ras-expressing thyroid cells progressed into S and G2 phases and evoked a checkpoint response characterized by the activation of ATR. Ras-stimulated ATR activity, as evidenced by Chk1 and p53 phosphorylation, was blocked by p27, suggesting that cell cycle progression triggers checkpoint activation, likely as a consequence of replication stress. These data reveal that Ras is capable of inducing a DNA damage response with characteristics similar to those reported in precancerous lesions. Our findings also suggest that the frequent mutational activation of Ras in thyroid tumors reflects the ability of Ras-expressing cells to bypass checkpoints and evade apoptosis rather than to simply increase proliferative potential.

Activation of platelet-activating factor (PAF) receptor stimulates nitric oxide (NO) release via protein kinase C-alpha in HEC-1B human endometrial epithelial cell line.

BackgroundImpairment of the fertility in the platelet-activating factor (PAF) receptor transgenic female mice suggests changes in PAF functions can influence uterine receptivity. We hypothesized that vasodilatory actions of PAF in the uterus was exerted by PAF-mediated nitric oxide (NO) release via activation of isoenzyme-specific protein kinase C (PKC).Materials and methodsInducible and endothelial NOS was shown by Reverse transcription polymerase chain reaction RT-PCR in cDNA synthesized from RNA extract of proliferative and secretory endometrium as well endometrial epithelial cell lines HEC-1B. The effect of WEB2170, NG-monomethyl-l-arginine (l-NMMA) and Ro 31-8220 on PAF mediated NO release by HEC-1B cell was determined. PAF induced translocation of PKCα hr. Before agonist stimulation, in HEC-1B cell and its antagonist effect by Ro 31-8220 was studied by Western immunoblot analysis. PKC isoenzyme regulated by PAF was determined in HEC-1B cell lysate by immunoprecipitation.ResultsPAF-evoked a rapid and concentration-dependent biphasic increase in total NO in human HEC-1B endometrial epithelial cell line [as measured by a Sievers NOA 280A NO Chemiluminescent Analyser.] This increase in NO release was attenuated by the PAF receptor antagonist, WEB2170. Inhibition of NO synthesis by NG-monomethyl-l-arginine produced marked dose-dependent attenuation of PAF-mediated NO release, indicating nitric oxide synthase (NOS) activation. PAF-mediated NO release was also inhibited by the PKC inhibitor Ro 31-8220 and by the removal of extracellular calcium, suggesting a dependency on PKC and calcium, respectively. RT-PCR analysis showed expression of inducible NOS and endothelial NOS in human endometrium, myometrium and HEC-1B cells. Western immunoblot analysis showed PKCα, βII and ι were the principal isozymes present in the HEC-1B cell line and normal endometrium, suggesting that both HEC-1B cells and normal endometrium have similar PKC isozymes. PAF induced the translocation of both PKCα and PKCι within the time frame of NO release. The translocation of PKCα, but not PKCι, was susceptible to inhibition by Ro 31-8220 that also inhibited PAF-evoked NO release, suggesting that PKCα is the principal isozyme involved in this process and that eNOS may be a substrate for PKCα. Kinase assays performed using immunoprecipitated PKCα showed that PAF (1 nM) activated PKCα that was inhibited by co-incubation with Ro 31-8220 and Ca2+-free medium.ConclusionsThis study demonstrates that PAF-stimulated NO release via PKCα in epithelial cells might regulate endometrial functions such as implantation and menstruation.

Protein kinase C inhibition induces DNA fragmentation in COLO 205 cells which is blocked by cysteine protease inhibition but not mediated through caspase-3

Enhancing apoptosis to remove abnormal cells has potential in reversing cancerous processes. Caspase-3 activation generally accompanies apoptosis and its substrates include enzymes responsible for DNA fragmentation and isozymes of protein kinase C (PKC). Recent data, however, question its obligatory role in apoptosis. We have examined whether modulation of PKC activity induces apoptosis in COLO 205 cells and the role of caspase-3. Proliferation ([3H]thymidine) and apoptosis (DNA fragmentation and FACS) of COLO 205 cells were measured in response to PKC activation and inhibition. Caspase-3 activity was assayed and the effects of its inhibition with Ac-DEVD-cmk, and the effect of other protease inhibitors, on apoptosis were determined. PKC activation and inhibition both reduced DNA synthesis and induced DNA fragmentation. As PKC inhibitors induced DNA fragmentation more rapidly than PKC activators and failed to block activator effects, we conclude that it is PKC down-regulation (i.e., inhibition) after activator exposure that mediates apoptosis. Increases in caspase-3 activity occurred during apoptosis but apoptosis was not blocked by caspase inhibition. By contrast, the cysteine protease inhibitor, E-64d, blocked apoptosis. Cysteine proteases not of the caspase family may either act more closely to the apoptotic process than caspases or lie on an alternative, more active pathway.

Role of EPAC in cAMP-Mediated Actions in Adrenocortical Cells.

Adrenocorticotropic hormone regulates adrenal steroidogenesis mainly via the intracellular signaling molecule cAMP. The effects of cAMP are principally relayed by activating protein kinase A (PKA) and the more recently discovered exchange proteins directly activated by cAMP 1 and 2 (EPAC1 and EPAC2). While the intracellular roles of PKA have been extensively studied in steroidogenic tissues, those of EPACs are only emerging. EPAC1 and EPAC2 are encoded by the genes RAPGEF3 and RAPGEF4, respectively. Whereas EPAC1 is ubiquitously expressed, the expression of EPAC2 is more restricted, and typically found in endocrine tissues. Alternative promoter usage of RAPGEF4 gives rise to three different isoforms of EPAC2 that vary in their N-termini (EPAC2A, EPAC2B, and EPAC2C) and that exhibit distinct expression patterns. EPAC2A is expressed in the brain and pancreas, EPAC2B in steroidogenic cells of the adrenal gland and testis, and EPAC2C has until now only been found in the liver. In this review, we discuss current knowledge on EPAC expression and function with focus on the known roles of EPAC in adrenal gland physiology.

POSTMan (POST-translational modification analysis), a software application for PTM discovery

Post‐translationally modified peptides present in low concentrations are often not selected for CID, resulting in no sequence information for these peptides. We have developed a software POSTMan (POST‐translational Modification analysis) allowing post‐translationally modified peptides to be targeted for fragmentation. The software aligns LC‐MS runs (MS1 data) between individual runs or within a single run and isolates pairs of peptides which differ by a user defined mass difference (post‐translationally modified peptides). The method was validated for acetylated peptides and allowed an assessment of even the basal protein phosphorylation of phenylalanine hydroxylase (PHA) in intact cells.

Gα12 binds to the N-terminal regulatory domain of p120ctn, and downregulates p120ctn tyrosine phosphorylation induced by Src family kinases via a RhoA independent mechanism

p120 Catenin (p120(ctn)) regulates cadherin stability, and thus facilitates strong cell-cell adhesion. Previously, we demonstrated that Gα(12) interacts with p120(ctn). In the present study, we have delineated a region of p120(ctn) that binds to Gα(12). We report that the N-terminal region of p120(ctn) (amino acids 1-346) is necessary and sufficient for the interaction. While the coiled-coiled domain and a charged region, comprising a.a 102-120, were found to be dispensable, amino acids 121-323 were required for p120(ctn) binding to Gα(12). This region harbors the phosphorylation domain of p120(ctn) and has been postulated as important for RhoA regulation. Downregulation of Src family kinase-induced tyrosine phosphorylation of p120(ctn) was observed in the presence of activated Gα(12). This down-regulation was triggered by three different Gα(12) mutants uncoupled from RhoA signalling. Furthermore, a dominant active form of RhoA did not reduce Src-induced phosphoryaltion of p120(ctn). In summary, our results suggest that Gα(12) binds to p120(ctn) and modulates its phosphorylation status through a Rho-independent mechanism. Gα(12) emerges as an important regulator of p120(ctn) function, and possibly of cadherin-mediated adhesion and/or cell motility.

PIK3CA exon9 mutations associate with reduced survival, and are highly concordant between matching primary tumors and metastases in endometrial cancer.

Mutations of the phosphoinositide-3-kinase (PI3K) catalytic subunit alpha gene (PIK3CA) are frequent in endometrial cancer. We sequenced exon9 and exon20 of PIK3CA in 280 primary endometrial cancers to assess the relationship with clinicopathologic variables, patient survival and associations with PIK3CA mRNA and phospho-AKT1 by gene expression and protein data, respectively. While PIK3CA mutations generally had no impact on survival, and were not associated with clinicopathological variables, patients with exon9 charge-changing mutations, providing a positive charge at the substituted amino acid residue, were associated with poor survival (p = 0.018). Furthermore, we characterized PIK3CA mutations in the metastatic setting, including 32 patients with matched primary tumors and metastases, and found a high level of concordance (85.7%; 6 out of 7 patients), suggesting limited heterogeneity. PIK3CA mRNA levels were increased in metastases compared to the primary tumors (p = 0.031), independent of PIK3CA mutation status, which rather associated with reduced PIK3CA mRNA expression. PIK3CA mutated tumors expressed higher p-AKT/AKT protein levels, both within primary (p < 0.001) and metastatic lesion (p = 0.010). Our results support the notion that the PI3K signaling pathway might be activated, both dependent- and independently of PIK3CA mutations, an aspect that should be considered when designing PIK3 pathway targeting strategies in endometrial cancer.