Yusufi, Ahad Noor Khan

Recent studies in cultured cells have provided evidence that a variety of pathobiologic stimuli, including high glucose, angiotensin II, and thromboxane A 2 , trigger a signaling pathway leading to autocrine induction of TGF-β1. TGF-β1 production through this pathway may profoundly affect cell growth, matrix synthesis, and response to injury. This study examines the role of autocrine versus exogenously added TGF-β1 in cellular proliferation and collagen IV production, critical targets of TGF-β1 signaling, using renal cells derived from TGF-β1 knockout (KO) animals or wild-type (WT) controls. Growth of WT and KO cells was assessed by cell counting and [ 3 H]thymidine uptake. Basal and TGF-β1-stimulated collagen production was assessed by Northern and Western blotting; transcriptional activity of the α1(IV) collagen gene was assessed by transient transfection analysis. KO cells grew at a faster rate than WT cells carefully matched for plating density and passage number. This increased growth rate was paralleled by increases in [ 3 H]thymidine uptake. KO cells expressed lower levels of the cell cycle inhibitors p21 and p27 than WT cells. KO cells failed to express TGF-β1, as expected. Basal TGF-β3 mRNA levels were higher in KO cells than in WT cells. WT cells expressed higher basal levels of TGF-β2 mRNA than KO cells. Basal α1(IV) and α2(IV) collagen mRNA and protein expression were significantly lower in KO cells than WT cells. Administration of exogenous TGF-β1 induced collagen IV production in both KO and WT cells. Although basal transcriptional activity of an α1(IV) collagen-CAT construct was lower in KO cells than WT cells, administration of exogenous TGF-β1 was associated with significant increases in transcriptional activity of this construct in both KO and WT cells. These studies provide evidence that autocrine production of TGF-β1 may play a critical role in regulation of growth and basal collagen IV production by renal tubular epithelial cells.

Signaling via release of Ca2+from intracellular stores is mediated by several systems, including the inositol 1,4,5-trisphosphate (IP3) and cADP-ribose (cADPR) pathway. We recently discovered a high capacity for cADPR synthesis in rat glomeruli and cultured mesangial cells (MC). We sought to determine whether 1) cADPR synthesis in MC is regulated by cytokines and hormones, 2) ryanodine receptors (RyRs) are expressed in MC, and 3) Ca2+is released through RyRs in response to cADPR. We found that ADP-ribosyl cyclase, a CD38-like enzyme that catalyzes cADPR synthesis, is upregulated in MC by tumor necrosis factor-α, interleukin-1β, and all- trans retinoic acid (atRA). [3H]ryanodine binds to microsomal fractions from MC with high affinity in a Ca2+-dependent manner; binding is enhanced by specific RyR agonists and blocked by ruthenium red and cADPR. Western blot analysis confirmed the presence of RyR in MC. Release of45Ca2+from MC microsomes was stimulated by cADPR; release was blocked by ruthenium red and 8-bromo-cADPR. ADPR (non-cyclic) was without effect. In MC, TNF-α and atRA amplified the increment of cytoplasmic Ca2+elicited by vasopressin. We conclude that MC possess elements of a novel ADP-ribosyl cyclase→cADPR→RyR→Ca2+-release signaling pathway subject to regulation by proinflammatory cytokines and steroid superfamily hormones.

The release of Ca2+ from intracellular stores is a fundamental element of signaling pathways involved in regulation of vascular tone, proliferation, apoptosis, and gene expression. Studies of sea urchin eggs have led to the identification of three functionally distinct Ca2+ signaling pathways triggered by IP3, cADPR, and NAADP. The coexistence and functional relevance of these distinct intracellular Ca2+ release systems has only been described in a few mammalian cell types. The purpose of this study was to determine whether the IP3, cADPR, and NAADP Ca2+ release systems coexist in smooth muscle cells (SMC) and to determine the specificity of these intracellular Ca2+ release pathways. Microsomes were prepared from rat aortic SMC (VSMC) and were loaded with 45Ca2+. cADPR, NAADP, and IP3 induced Ca2+ release from VSMC microsomes in a dose-dependent fashion. Heparin blocked only IP3-mediated Ca2+ release, whereas the ryanodine channel inhibitors 8-Br-cADPR and ruthenium red blocked only cADPR-induced Ca2+ release. Nifedipine, an L-type Ca2+ channel blocker, inhibited NAADP elicited Ca2+ release, but had no effect on IP3- or cADPR-mediated Ca2+ release. An increase in pH from 7.2 to 8.2 inhibited cADPR-mediated Ca2+ release, but had no effect on IP3- or NAADP-induced Ca2+ release. By RT-PCR, VSMC expressed ryanodine receptor types 1, 2, and 3. Ca2+-dependent binding of [3H]-ryanodine to VSMC microsomes was enhanced by the ryanodine receptor agonists 4-chloro-methyl-phenol (CMP) and caffeine, but was inhibited by ruthenium red and cADPR. We conclude that VSMC possess at least three functionally distinct pathways that promote intracellular Ca2+ release. IP3-, cADPR-, and NAADP-induced intracellular Ca2+ release may play a critical role in the maladaptive responses of VSMC to environmental stimuli that are characteristically associated with hypertension and/or atherogenesis.

Nicotinic acid–adenine dinucleotide phosphate (NAADP), a molecule derived from β-NADP, has been shown to promote intracellular calcium release in sea urchin eggs. However, there is little information regarding the role of NAADP in the regulation of intracellular calcium fluxes in mammalian cells. We found recently that several mammalian tissues have a high capacity for NAADP synthesis, as assessed by sea urchin egg bioassay. To determine the functional significance of NAADP production by mammalian tissues, we sought to determine whether NAADP is capable of inducing calcium release from microsomes prepared from cultured cells. We found that NAADP, but not β-NADP, activates a specific microsomal calcium release system in mesangial cells isolated from rat kidney; NAADP was without effect in renal tubular epithelial cells. NAADP-induced calcium release is not affected by inhibitors of the inositol 1,4,5-trisphosphate or ryanodine channels. However, NAADP-elicited calcium release was inhibited by L-type calcium channel blockers and by alkaline phosphatase treatment of NAADP. NAADP also promotes specific microsomal calcium release in rat vascular smooth muscle cells, cardiac myocytes, fibroblasts and a human leukaemia cell line, indicating that the capacity for NAADP-induced calcium release is widespread in mammalian cells. We propose that NAADP may be an important regulator of intracellular calcium in many mammalian tissues.