Involvement of myristoylated alanine-rich c kinase substrate (MARCKS) protein in prostaglandin F2α- induced secretion of oxytocin by the bovine corpus luteum

Prostaglandin F₂α (PGF₂α), a stimulatory hormone of luteal oxytocin secretion, is known to activate protein kinase C (PKC); however, the intracellular signals that promote exocytosis of oxytocin remain to be elucidated. Myristoylated alanine-rich C kinase substrate (MARCKS), a protein specifically phosphorylated by PKC, crosslinks actin filaments associated with the inner leaflet of the plasma membrane. Studies were conducted to determine the role of the MARCKS protein in exocytosis of PGF₂α-induced bovine luteal oxytocin. Experiment 1 was conducted to examine the regulatory aspects and localization of MARCKS in the bovine corpus luteum (CL) in response to PGF₂α stimulation. Luteal cells were incubated with [32PJ-orthophosphate and stimulated with ethanol, PGF₂α, TPA and A23187. Treatments with PGF₂α, TPA and A23187 resulted in increased phosphorylation of MARCKS. Subsequently, heifers were injected with either saline or PGF2a and CL was collected 5 mm after treatments. Western blotting of the luteal samples indicated that PGF2- induced translocation of MARCKS from the membrane to cytoplasm within 5 mm. The aim of experiment 2 was to identify the specific PKC isoform activated by PGF₂α that phosphorylates MARCKS protein. Using isoform specific inhibitory peptides and polyclonal antibodies, it was observed that PKCx was the mediator of PGF₂α-induced phosphorylation of MARCKS. Experiment 3 was conducted to determine if phosphorylation dependent movement of MARCKS is related to the disruption of the actin cortex and exocytosis of oxytocin. For this purpose, luteal cells were transfected with green fluorescent protein (GFP) conjugated MARCKS cDNA constructs. Expression of MARCKS-GFP was observed within 18 hr after transfection. Cells were then treated with vehicle or PGF₂α and fixed with 4% paraformaldehyde. Cells were subjected to oxytocin antibody+rhodamine labeled secondary antibody and phalloidin, a specific marker of the actin cortex. Upon PGF₂α treatment, the wt MARCKS-GFP translocated from membrane to cytoplasm and actin filaments shortened. Oxytocin granules were mobilized towards the membrane from a paranuclear location. Phosphorylation and myristoylation mutant MARCKS-GFPs were not affected by PG₂α stimulation of cells and no exocytosis of oxytocin occurred. In summary, the research suggests that PGF₂α-induced PKCα mediated phosphorylation of MARCKS is closely correlated with exocytosis of oxytocin by the bovine CL

Functional characterization of the S41Y (C2755A) polymorphism of tryptophan hydroxylase 2

Human TPH2 (hTPH2) catalyzes the rate-limiting step in CNS serotonin biosynthesis. We characterized a single-nucleotide polymorphism (C2755A) in the hTPH2 gene that substitutes tyrosine for serine at position 41 in the regulatory domain of the enzyme. This polymorphism is associated with bipolar disorder and peripartum depression in a Chinese population. Recombinant h TPH2 human proteins were expressed in bacteria and also stably expressed in PC12 cells. Following bacterial expression and purification, the tyrosine for serine substitution at position 41 (S41Y) polymorphic enzyme displayed increased Vmax with unchanged Km values. By contrast, enzyme stability was decreased in vitro from 32 min to 4 min (37°C) for the S41Y enzyme (as compared to the wild-type enzyme). The S41Y polymorphism decreased cyclic AMP-dependent protein kinase A-mediated phosphorylation ~ 50% relative to wild-type hTPH2, suggesting that the S41Y mutation may disrupt the post-translational regulation of this enzyme. Transfected PC12 cells expressed hTPH2 mRNA, active protein, and synthesized and released serotonin. Paradoxically, while S41Y-transfected PC12 cells expressed higher levels of hTPH2 than wild type, they synthesized less serotonin. These findings suggest a modified regulation of the S41Y gene variant leading to altered regulation and reduced neurotransmitter synthesis that may contribute to association of the polymorphism with bipolar disorder and depression. © 2014 International Society for Neurochemistry

Robust activation of the human but not mouse telomerase gene during the induction of pluripotency

Pluripotent stem cells (PSCs) express telomerase and have unlimited proliferative potential. To study telomerase activation during reprogramming, 3 classes of embryonic stem cell (ESC)-like clones were isolated from mouse fibroblasts containing a transgenic hTERT reporter. Class I expressed few pluripotency markers, whereas class II contained many, but not Oct4, Nanog, and Sox2. Neither class of cells differentiated efficiently. Class III cells, the fully reprogrammed induced PSCs (iPSCs), expressed all pluripotency markers, formed teratomas indistinguishable from those of mESCs, and underwent efficient osteogenic differentiation in vitro . Interestingly, whereas the endogenous mTERT gene expression was only moderately increased during reprogramming, the hTERT promoter was strongly activated in class II cells and was further elevated in class III cells. Treatment of class II cells with chemical inhibitors of MEKs and glycogen synthase kinase 3 resulted in their further reprogramming into class III cells, accompanied by a strong activation of hTERT promoter. In reprogrammed human cells, the endogenous telomerase level, although variable among different clones, was dramatically elevated. Only in cells with the highest telomerase were telomeres restored to the lengths in hESCs. Our data, for the first time, demonstrated that the hTERT promoter was strongly activated in discrete steps, revealing a critical difference in human and mouse cell reprogramming. Because telomere elongation is crucial for self-renewal of hPSCs and replicative aging of their differentiated progeny, these findings have important implications in the generation and applications of iPSCs.—Mathew, R., Jia, W., Sharma, A., Zhao, Y., Clarke, L. E., Cheng, X., Wang, H., Salli, U., Vrana, K. E., Robertson, G. P., Zhu, J., Wang, S. Robust activation of the human but not mouse telomerase gene during the induction of pluripotency