Calcium/calmodulin-dependent kinases can regulate the TSH expression in the rat pituitary.

PURPOSE: The endocrine secretion of TSH is a finely orchestrated process controlled by the thyrotropin-releasing hormone (TRH). Its homeostasis and signaling rely on many calcium-binding proteins belonging to the "EF-hand" protein family. The Ca2+/calmodulin (CaM) complex is associated with Ca2+/CaM-dependent kinases (Ca2+/CaMK). We have investigated Ca2+/CaMK expression and regulation in the rat pituitary. METHODS: The expression of CaMKII and CaMKIV in rat anterior pituitary cells was shown by immunohistochemistry. Cultured anterior pituitary cells were stimulated by TRH in the presence and absence of KN93, the pharmacological inhibitor of CaMKII and CaMKIV. Western blotting was then used to measure the expression of these kinases and of the cAMP response element-binding protein (CREB). TSH production was measured by RIA after time-dependent stimulation with TRH. Cells were infected with a lentiviral construct coding for CaMKIV followed by measurement of CREB phosphorylation and TSH. RESULTS: Our study shows that two CaM kinases, CaMKII and CaMKII, are expressed in rat pituitary cells and their phosphorylation in response to TRH occurs at different time points, with CaMKIV being activated earlier than CaMKII. TRH induces CREB phosphorylation through the activity of both CaMKII and CaMKIV. The activation of CREB increases TSH gene expression. CaMKIV induces CREB phosphorylation while its dominant negative and KN93 exert the opposite effects. CONCLUSION: Our data indicate that the expression of Ca2+/CaMK in rat anterior pituitary are correlated to the role of CREB in the genetic regulation of TSH, and that TRH stimulation activates CaMKIV, which in turn phosphorylates CREB. This phosphorylation is linked to the production of thyrotropin

Calcium-binding protein and some neuropeptides in the retina of Octopus vulgaris: A morpho-histochemical study.

The existence of both calcium-binding proteins (CBPs) and neuropeptides in the retina and brain of various species of vertebrates and invertebrates is well documented. Octopus retina is particularly interesting because it represents a case of convergent evolution. The aim of this study was to characterize the distribution of two CBPs, calretinin and calbindin, in Octopus retina using morphology, in situ hybridization, immunocytochemistry and Western blot. Calretinin-like immunoreactivity was found in the photoreceptor cells, but unexpectedly also in the supporting cells. In situ hybridization and Western blot analysis confirmed these results. No immunoreactivity was found for calbindin. Two neuropeptides, Substance P and calcitonin gene-related peptide (CGRP), as well as neurofilament protein and glial fibrillary acidic protein were also localized in the Octopus retina by immunocytochemistry. Our work provides new insights about calcium-binding proteins and neuropeptide distribution in Octopus retina and suggests a functional role for calretinin, a highly conserved protein, in visual signal transduction of cephalopods

Copper/Zinc-Superoxide Dismutase in Human Epidermis: An Immunochemical Study.

The localization of copper and zinc-superoxide dismutase in normal and neoplastic human skin was examined with immunochemical techniques. Skin samples were taken from males and females of different ages, UV exposed and non-exposed areas and basal-/spino-cellular carcinomas. The enzyme was localized diffusely in the cytoplasm and was also found in the nuclei of epidermal cells, endothelial cells and other dermis cell types. The dismutase content in the epidermis was higher in males than females, UV-exposed than non-exposed and young than old people. In the tumors, the enzyme content of the superficial epidermal layers was higher than in the deep tumoral epithelial cells. These data suggest that the localization of Cu, Zn-SOD in skin tissues reflects the gender and age of the subject, the cell types and their normal or diseased state. Further studies based on the investigation of systemic changes of this enzyme in physiological and pathological epidermis could provide additional information on tumor cell generation

Non-canonical role of PDK1 as a negative regulator of apoptosis through macromolecular complexes assembly at the ER-mitochondria interface in oncogene-driven NSCLC

Here, we tested whether co-targeting of glucose metabolism and oncogene drivers may enhance tumor response to tyrosine kinase inhibitors (TKIs) in NSCLC. To this end, pyruvate dehy-drogenase kinase 1 (PDK1) was stably downregulated in oncogene-driven NSCLC cell lines exposed or not to TKIs. H1993 and H1975 cells were stably transfected with scrambled (shCTRL) or PDK1-targeted (shPDK1) shRNA and then treated with MET inhibitor crizotinib (1 µM), double mutant EGFRL858R/T790M inhibitor WZ4002 (1 µM) or vehicle for 48 h. The effects of PDK1 knockdown on glucose metabolism and apoptosis were evaluated in untreated and TKI-treated cells. PDK1 knockdown alone did not cause significant changes in glycolytic cascade, ATP production and glucose consumption, but it enhanced maximal respiration in shPDK1 cells when compared to controls. When combined with TKI treatment, PDK1 downregulation caused a strong enhancement of OXPHOS and a marked reduction in key glycolytic enzymes. Furthermore, increased levels of apoptotic markers were found in shPDK1 cells as compared to shCTRL cells after treatment with TKIs. Co-immunoprecipitation studies showed that PDK1 interacts with PKM2, Bcl-2 and Bcl-xL, forming macromolecular complexes at the ER–mitochondria interface. Our findings showed that downreg-ulation of PDK1 is able to potentiate the effects of TKIs through the disruption of macromolecular complexes involving PKM2, Bcl-2 and Bcl-xL