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Gαq (Gene GNAQ) plays a major role in platelet signal transduction but little is known regarding its transcriptional regulation. We studied Gαq promoter activity using luciferase reporter gene assays in human erythroleukemia (HEL) cells treated with 10 nM of phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic (MK) transformation. RT-PCR analysis of HEL cell RNA revealed that Gαq mRNA was relatively low in untreated cells and it increased after PMA treatment with a peak at 5 h. Immunoblot analysis of HEL lysates showed enhanced Gαq expression with PMA. Luciferase reporter gene studies on full length construct (upto −1116 bp from ATG) and its serial 5′ truncations revealed a negative regulatory site at −238/−202 and two positive sites at −203/−138 and −1116/−731. In the region −238/−202 consensus sites were noted for two transcription factors PU.1 and GATA-1 that are known to regulate several megakaryocytic genes. Deletions of these sites alone or together revealed no change in the transcriptional activity of the gene in reporter studies. The positive region −203/−138 contained two overlapping Sp1/AP-2/EGR-1 consensus sites at −202/−189 and −164/−150. Gel shift studies were performed on oligonucleotides 1 (−203/−175) and 2 (−174/−152) using HEL cell extracts. Protein binding occured with Gαq oligonucleotides 1 and 2, which was competed with excess unlabeled oligos but not by unlabeled Sp1 or AP-2 consensus oligos. Supershift assay using antibody against Sp1 revealed neither competition nor supershift, suggesting that Sp1 does not bind to these oligonucleotides. No protein binding was noted when Gαq oligos 1 or 2 were incubated with extracts known to contain Sp1 or AP-2. These results indicate that Sp1 and AP-2 do not bind to the Gαq oligonucleotide regions. Protein binding to oligonucleotides 1 and 2 was abolished by excess unlabeled consensus EGR-1 oligo, and by immunodepletion of the EGR-1 protein from the nuclear extract with anti-EGR-1 antibody. These experiments reveal that EGR-1 binds to both Gαq oligonucleotides −203/−175 and −174/−152. In luciferase reporter studies mutations in EGR-1 sites present in both oligonucleotides 1 and 2 markedly decreased gene activity indicating functional relevance. In further studies, reduction in endogenous EGR-1 expression with antisense oligonucleotide to EGR-1 inhibited PMA induced Gαq transcription and protein in HEL cells. Lastly, EGR-1 deficient mouse platelets also showed ~50% reduction in the Gαq protein (immunoblotting) relative to wild type platelets. These studies suggest that Gαq gene is regulated during PMA induced differentiation by EGR-1, a transcription factor that regulates a wide array of genes involved in cellular proliferation, differentiation, and apoptosis, and in vascular response to injury and atherosclerosis.

A model system to study the biochemical mechanism of follicular atresia in rats [Dhanasekaran et al. 1983] was characterized using histological and biochemical correlates. PMSG and PMSG antiserum (a/s) was used to induce the follicular growth and atresia of preovulatory follicles. Ovarian histology during these PMSG and PMSG a/s - treatment periods was recorded under a light microscope. An analysis of lysosomal enzyme cathepsin-D activity of granulosa cells (GC) from similarly treated ovaries showed that there was a reduction in cathepsin-D activity during the histologically observable follicular growth; and there was an increase in cathepsin-D activity during atresia. The increase in cathepsin-D activity also showed an inverse correlation with the general anabolic activity of the GC as demonstrated here, by a reduction in 3H-leucine incorporation activity. An analysis of other gonadotropin-responsive cells for the presence of such hormone sensitive lysosomal machinery, only corpora lutea (CL) and GC showed in the cathepsin-D activity upon treatment with 15 IU of PMSG. The results suggests the existence of a common gonadotropin regulated lysosomal machinery in cells endowed with a degenerative pathway of "programmed cell death". More importantly the results establish the validity of using lysosomal enzyme cathepsin-D as a biochemical marker, for hitherto morphologically and endocrinologically studied cellular degenerative process of follicular atresia.

International audience; Galpha13, the alpha-subunit of the heterotrimeric G protein G13, has been shown to stimulate cell migration in addition to inducing oncogenic transformation. Cta, a Drosophila ortholog of G13, has been shown to be critical for cell migration leading to the ventral furrow formation in Drosophila embryos. Loss of Galpha13 has been shown to disrupt cell migration associated with angiogenesis in developing mouse embryos. Whereas these observations point to the vital role of G13-orthologs in regulating cell migration, widely across the species barrier, the mechanism by which Galpha13 couples to cytoskeleton and cell migration is largely unknown. Here we show that Galpha13 physically interacts with Hax-1, a cytoskeleton-associated, cortactin-interacting intracellular protein, and this interaction is required for Galpha13-stimulated cell migration. Hax-1 interaction is specific to Galpha13, and this interaction is more pronounced with the mutationally or functionally activated form of Galpha13 as compared with the wild-type Galpha13. Expression of Hax-1 reduces the formation of actin stress fibers and focal adhesion complexes in Galpha13-expressing NIH3T3 cells. Coexpression of Hax-1 also attenuates Galpha(13)-stimulated activity of Rho while potentiating Galpha13-stimulated activity of Rac. The presence of a quadnary complex consisting of Galpha13, Hax-1, Rac, and cortactin indicates the role of Hax-1 in tethering Galpha13 to the cytoskeletal component(s) involved in cell movement. Whereas the expression of Hax-1 potentiates Galpha13-mediated cell movement, silencing of endogenous Hax-1 with Hax-1-specific small interfering RNAs drastically reduces Galpha13-mediated cell migration. These findings, along with the observation that Hax-1 is overexpressed in metastatic tumors and tumor cell lines, suggest a novel role for the association of oncogenic Galpha13 and Hax-1 in tumor metastasis.