Walker, Henry J.

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.

Polycystic kidney diseases (PKD) are characterized by excessive proliferation of renal tubular epithelial cells, development of fluid-filled cysts, and progressive renal insufficiency. cAMP inhibits proliferation of normal renal tubular epithelial cells but stimulates proliferation of renal tubular epithelial cells derived from patients with PKD. Madin-Darby canine kidney (MDCK) epithelial cells, which are widely used as an in vitro model of cystogenesis, also proliferate in response to cAMP. Intracellular cAMP levels are tightly regulated by phosphodiesterases (PDE). Isoform-specific PDE inhibitors have been developed as therapeutic agents to regulate signaling pathways directed by cAMP. In other renal cell types, we have previously demonstrated that cAMP is hydrolyzed by PDE3 and PDE4, but only PDE3 inhibitors suppress proliferation by inhibiting Raf-1 activity (Cheng J, Thompson MA, Walker HJ, Gray CE, Diaz Encarnacion MM, Warner GM, Grande JP. Am J Physiol Renal Physiol 287:F940-F953, 2004.) A potential role for PDE isoform(s) in cAMP-mediated proliferation of MDCK cells has not previously been established. Similar to what we have previously found in several other renal cell types, cAMP hydrolysis in MDCK cells is directed primarily by PDE4 (85% of total activity) and PDE3 (15% of total activity). PDE4 inhibitors are more effective than PDE3 inhibitors in increasing intracellular cAMP levels in MDCK cells. However, only PDE3 inhibitors, and not PDE4 inhibitors, stimulate mitogenesis of MDCK cells. PDE3 but not PDE4 inhibitors activate B-Raf but not Raf-1, as assessed by an in vitro kinase assay. PDE3 but not PDE4 inhibitors activate the ERK pathway and activate cyclins D and E, as assessed by histone H1 kinase assay. We conclude that mitogenesis of MDCK cells is regulated by a functionally compartmentalized intracellular cAMP pool directed by PDE3. Pharmacologic agents that stimulate PDE3 activity may provide the basis for new therapies directed toward reducing cystogenesis in patients with PKD.