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Receptor-operated Ca 2ϩ entry (ROCE) via transient receptor potential canonical channel 6 (TRPC6) is important machinery for an increase in intracellular Ca 2ϩ concentration triggered by the activation of G q protein-coupled receptors. TRPC6 is phosphorylated by various protein kinases including protein kinase A (PKA). However, the regulation of TRPC6 activity by PKA is still controversial. The purpose of this study was to elucidate the role of adenylate cyclase/cAMP/PKA signaling pathway in the regulation of G q protein-coupled endothelin type A receptor (ET A R)-mediated ROCE via TRPC6. For this purpose, human embryonic kidney 293 (HEK293) cells stably coexpressing human ET A R and TRPC6 (wild type) or its mutants possessing a single point mutation of putative phosphorylation sites for PKA were used to analyze ROCE and amino acids responsible for PKA-mediated phosphorylation of TRPC6. Ca 2ϩ measurements with thapsigargin-induced Ca 2ϩ -depletion/Ca 2ϩ -restoration protocol to estimate ROCE showed that the stimulation of ET A R induced marked ROCE in HEK293 cells expressing TRPC6 compared with control cells. The ROCE was inhibited by forskolin and papaverine to activate the cAMP/PKA pathway, whereas it was potentiated by Rp-8-bromoadenosinecAMP sodium salt, a PKA inhibitor. The inhibitory effects of forskolin and papaverine were partially cancelled by replacing Ser28 (TRPC6 S28A ) but not Thr69 (TRPC6 T69A ) of TRPC6 with alanine. In vitro kinase assay with Phos-tag biotin to determine the phosphorylation level of TRPC6 revealed that wild-type and mutant (TRPC6 S28A and TRPC6 T69A ) TRPC6 proteins were phosphorylated by PKA, but the phosphorylation level of these mutants was lower (approximately 50%) than that of wild type. These results suggest that TRPC6 is negatively regulated by the PKA-mediated phosphorylation of Ser28 but not Thr69.

Although the mammalian intestinal epithelium manifests robust regenerative capacity after various cytotoxic injuries, the underlying mechanism has remained unclear. Here we identify the cyclin-dependent kinase inhibitor p57 as a specific marker for a quiescent cell population located around the +4 position of intestinal crypts. Lineage tracing reveals that the p57+ cells serve as enteroendocrine/tuft cell precursors under normal conditions but dedifferentiate and act as facultative stem cells to support regeneration after injury. Single-cell transcriptomics analysis shows that the p57+ cells undergo a dynamic reprogramming process after injury that is characterized by fetal-like conversion and metaplasia-like transformation. Population-level analysis also detects such spatiotemporal reprogramming widely in other differentiated cell types. In intestinal adenoma, p57+ cells manifest homeostatic stem cell activity, in the context of constitutively activated spatiotemporal reprogramming. Our results highlight a pronounced plasticity of the intestinal epithelium that supports maintenance of tissue integrity in normal and neoplastic contexts.

Abstract. The purpose of this study is to identify transient receptor potential canonical (TRPC) channels responsible for receptor-operated Ca 2+ entry (ROCE) triggered by activation of endothelin type A receptor (ET A R) and to clarify the importance of calmodulin (CaM) / inositol 1,4,5-trisphosphate (IP 3 ) receptor binding (CIRB) domain at the C terminus of TRPC channels in ET A Ractivated channel regulation. In HEK293 cells coexpressing ET A R and one of seven TRPC isoforms, ET A R stimulation induced ROCE through TRPC3, TRPC5, TRPC6, and TRPC7. The TRPC3-and TRPC6-mediated ROCE was inhibited by selective inhibitors of G q protein, phospholipase C (PLC), and CaM. The CIRB domain deletion mutants of TRPC3 and TRPC6 failed to induce ET A R-mediated ROCE. Either deletion of the CIRB domain or pharmacological inhibition of CaM did not inhibit the targeting of these channels to the plasma membrane. These results suggest that 1) TRPC3, TRPC5, TRPC6, and TRPC7 can function as ET A R-operated Ca 2+ channels; 2) G q protein, PLC, and CaM are involved in TRPC3-and TRPC6-mediated ROCE; 3) ET A R-mediated activation of TRPC3 and TRPC6 requires the CIRB domain; and 4) abolition of ET A R-induced ROCE by CIRB domain deletion and CaM inhibition is due to loss of CaM binding to the channels but not loss of cell surface TRPC3 and TRPC6.

BACKGROUND AND PURPOSEEndothelin-1 (ET-1) reduces insulin-stimulated glucose uptake in skeletal muscle, inducing insulin resistance. Here, we have determined the molecular mechanisms underlying negative regulation by ET-1 of insulin signalling. EXPERIMENTAL APPROACHWe used the rat L6 skeletal muscle cells fully differentiated into myotubes. Changes in the phosphorylation of Akt was assessed by Western blotting. Effects of ET-1 on insulin-stimulated glucose uptake was assessed withThe C-terminus region of GPCR kinase 2 (GRK2-ct), a dominant negative GRK2, was overexpressed in L6 cells using adenovirusmediated gene transfer. GRK2 expression was suppressed by transfection of the corresponding short-interfering RNA (siRNA). KEY RESULTSIn L6 myotubes, insulin elicited sustained Akt phosphorylation at Thr 308 and Ser 473 , which was suppressed by ET-1. The inhibitory effects of ET-1 were prevented by treatment with a selective ET A receptor antagonist and a G q protein inhibitor, overexpression of GRK2-ct and knockdown of GRK2. Insulin increased [ 3 H]2-DG uptake rate in a concentration-dependent manner. ET-1 noncompetitively antagonized insulin-stimulated [ 3 H]2-DG uptake. Blockade of ET A receptors, overexpression of GRK2-ct and knockdown of GRK2 prevented the ET-1-induced suppression of insulin-stimulated [ 3 H]2-DG uptake. In L6 myotubes overexpressing FLAG-tagged GRK2, ET-1 facilitated the interaction of endogenous Akt with FLAG-GRK2. CONCLUSIONS AND IMPLICATIONSActivation of ET A receptors with ET-1 suppressed insulin-induced Akt phosphorylation at Thr 308 and Ser 473 and [ 3 H]2-DG uptake in a GRK2-dependent manner in skeletal muscle cells. These findings suggest that ET A receptors and GRK2 are potential targets for overcoming insulin resistance.