Anti-CGB monoclonal antibody, clone DH15 + DH16

Abnormal trophoblast function is associated with fetal growth restriction (FGR). The JAK-STAT pathway is one of the principal signalling mechanisms by which cytokines and growth factors modulate cell proliferation, differentiation, cell migration and apoptosis. The expression of placental JAK-STAT genes in human idiopathic FGR is unknown. In this study, we propose the hypothesis that JAK-STAT pathway genes are differentially expressed in idiopathic FGR-affected pregnancies and contribute to abnormal feto-placental growth by modulating the expression of the amino acid transporter SNAT2, differentiation marker CGB/human chorionic gonadotrophin beta-subunit (beta-hCG) and apoptosis markers caspases 3 and 8, and TP53. Expression profiling of FGR-affected placentae revealed that mRNA levels of STAT3, STAT2 and STAT5B decreased by 69, 52 and 50%, respectively, compared with gestational-age-matched controls. Further validation by real-time PCR and immunoblotting confirmed significantly lower STAT3 mRNA and STAT3 protein (total and phosphorylated) levels in FGR placentae. STAT3 protein was localised to the syncytiotrophoblast (ST) in both FGR and control placentae. ST differentiation was modelled by in vitro differentiation of primary villous trophoblast cells from first-trimester and term placentae, and by treating choriocarcinoma-derived BeWo cells with forskolin in cell culture. Differentiation in these models was associated with increased STAT3 mRNA and protein levels. In BeWo cells treated with siRNA targeting STAT3, the mRNA and protein levels of CGB/beta-hCG, caspases 3 and 8, and TP53 were significantly increased, while that of SNAT2 was significantly decreased compared with the negative control siRNA. In conclusion, we report that decreased STAT3 expression in placentae may contribute to abnormal trophoblast function in idiopathic FGR-affected pregnancies.

Neuropeptide precursors synthesized at the rough endoplasmic reticulum are transported and sorted at the trans-Golgi network (TGN) to the granules of the regulated secretory pathway (RSP) of neuroendocrine cells. They are then processed into active peptides and stored in large dense-core granules (LDCGs) until secreted upon stimulation. We have studied the regulation of biogenesis of the LDCGs and the mechanism by which neuropeptide precursors, such as pro-opiomelanocortin (POMC), are sorted into these LDCGs of the RSP in neuroendocrine and endocrine cells. We provide evidence that chromogranin A (CgA), one of the most abundant acidic glycoproteins ubiquitously present in neuroendocrine/endocrine cells, plays an important role in the regulation of LDCG biogenesis. Specific depletion of CgA expression by antisense RNAs in PC12 cells led to a profound loss of secretory granule formation. Exogenously expressed POW was neither stored nor secreted in a regulated manner in these CgA-deficient PC12 cells. Overexpression of CgA in a CgA- and LDCG-deficient endocrine cell line, 6T3, restored regulated secretion of transfected POW and the presence of immunoreactive CgA at the tips of the processes of these cells. Unlike CgA, CgB, another granin protein, could not substitute for the role of CgA in regulating LDCG biogenesis. Thus, we conclude that CgA is a key player in the regulation of the biogenesis of LDCGs in neuroendocrine cells. To examine the mechanism of sorting POW to the LDCGs, we carried out site-directed mutagenesis, transfected the POW mutants into PC12 cells, and assayed for regulated secretion. Our previous molecular modeling studies predicted a three-dimensional sorting motif in POW that can bind to a sorting receptor, membrane carboxypeptidase E (CPE). The sorting signal consists of four conserved residues at the N-terminal loop structure of POMC: two acidic residues and two hydrophobic residues. The two acidic residues were predicted to bind to a domain on CPE (CPE254-273) containing two basic residues (R255 and K260) to effect sorting into immature secretory granules. Site-directed mutagenesis of the motif on POW resulted in accumulation of the mutant in the Golgi, as well as high basal secretion, indicating that the mutant POW was inefficiently sorted to the RSP These results support the model that POW is actively sorted to the RSP granules for processing and secretion by a sorting signal-mediated mechanism.