TGF-β Concentration Specifies Differential Signaling Profiles of Growth Arrest/Differentiation and Apoptosis in Podocytes

Abstract

Podocyte depletion occurs in most progressive glomerular diseases and is thought to result from podocyte loss while the remaining podocytes are unable to proliferate. The underlying mechanisms for podocyte growth arrest/differentiation and depletion remain poorly understood but may involve TGF-␤, which is typically upregulated in injured glomeruli. The TGF-␤ are multifunctional cytokines that regulate growth, differentiation, and apoptosis in most cells. Determinants of functional specificity of TGF-␤ signaling in cell-cycle control and apoptosis remain poorly understood. Using a unique system of conditionally immortalized podocytes, it is demonstrated that autocrine TGF-␤2 induces G0/G1 arrest and differentiation under nonpermissive culture through Smad3-dependent induction of the cyclin-dependent kinase inhibitor p15 Ink4b (Cdkn2b). When exposed to recombinant TGF-␤1 (or TGF-␤2), nonpermissive culture podocytes switch to G2/M arrest and apoptosis, selectively at advanced TGF-␤ concentrations and specifically in association with suppression of Cdkn2b and activation of proapoptotic p38 mitogen-activated protein kinase. Thus, distinct signaling profiles activated in a concentrationdependent manner by TGF-␤ were identified. Autocrine TGF-␤2/Smad3/Cdkn2b signaling in podocytes specifies G0/G1 arrest associated with podocyte differentiation, whereas increasing TGF-␤ concentrations beyond a critical threshold induces G2/M block and apoptosis associated with selective p38 mitogen-activated protein kinase activation and with suppression of Cdkn2b. In summary, the results suggest a new functional requirement of TGF-␤2 in growth arrest and differentiation of murine podocytes in vitro and demonstrate that a critical TGF-␤ concentration threshold may specify a molecular switch to proapoptotic signaling profiles and apoptosis.

TβRI Independently Activates Smad- and CD2AP-Dependent Pathways in Podocytes

“…4 Levels of cyclin D1, a critical promoter of early cell-cycle progression in G1/S phase, were considerably increased in DKO and absent in CKO, compared with WT podocytes ( Figure 4E). Protein and mRNA levels of the cyclin-dependent kinase inhibitor (cdki) p15…”

“…Autocrine and paracrine TGF-␤ induces G0/G1 arrest in podocytes in a Smad3-dependent manner. 4 To examine the relative roles of Smad2/3 and CD2AP in baseline and TGF-␤-stimulated growth regulation of podocytes, we quantified growth rates of WT, DKO, and CKO podocytes using tetrazolium salt WST-1-based proliferation assays as described previously. 28 Growth of untreated CKO cells was significantly reduced compared with WT podocytes, whereas untreated DKO podocytes proliferated at a significantly increased rate ( Figure 4A).…”

“…1,2 Similar to other epithelial cell types, TGF-␤ stimulation induces apoptosis, growth inhibition, or differentiation in podocytes. [3][4][5] TGF-␤1 transgenic mice (TGF-␤1tg) are characterized by podocyte apoptosis at the onset of proteinuria and progressive glomerulosclerosis associated with progressive podocyte depletion. 3 Podocyte depletion is considered a hallmark of glomerular diseases characterized by segmental or global glomerulosclerosis.…”

“…Although these proapoptotic responses to TGF-␤ are largely mediated by Smaddependent regulation of proapoptotic target genes, 14,15 TGF-␤ also activates prosurvival pathways such as mitogen-activated protein kinases (MAPKs) 16,17 and phosphoinositide-3 kinases (PI3Ks). 18,19 Previously, we showed that Smad3 mediates apoptotic signaling of TGF-␤ via the p38 MAPK pathway in podocytes 4 and reported that the adaptor molecule CD2-associated protein (CD2AP) is required for rapid, early activation of the antiapoptotic PI3K/AKT pathway in podocytes 20 ; however, it is not known whether or how Smad proteins and CD2AP interact or how PI3K/AKT is linked with TGF-␤R complexes.…”

Role of MKK3–p38 MAPK signalling in the development of type 2 diabetes and renal injury in obese db/db mice

“…In addition, p38 signalling can contribute to proinflammatory and profibrotic responses. Activation of p38 enhances production of monocyte chemoattractant protein-1 (MCP-1) by vascular endothelial cells [23], induces local angiotensinogen production in rat tubular cells [17], stimulates both TGF-β-induced fibronectin accumulation in renal interstitial fibroblasts [24] and collagen production in mouse mesangial cells [25], increases Tgf-β1 (also known as Tgfb1) expression in renal tubular cells [26] and promotes synthesis of vascular endothelial growth factor induced by angiotensin II [27,28]. Studies have also shown that p38 signalling mediates both tubular hypertrophy induced by high glucose [26] and transactivation of the epidermal growth factor receptor required for dedifferentiation of proximal tubular epithelial cells following oxidant injury [29].…”