Leof, Edward B.

ABSTRACT Transforming growth factor‐β (TGF‐β) is the single most important cytokine promoting renal fibrogenesis. p21‐activated kinase‐2 (PAK2) and activa‐ tion of abelson nonreceptor tyrosine kinase (c‐abl) have been shown recently to be smad‐independent, fibro‐ blast‐specific targets downstream of the activated TGF‐β receptor. In the current study we show that in cultured NRK49F‐renal fibroblasts (but not in tubular or mesangial cells) TGF‐β similarly activates PAK2 as well as c‐abl and induces cell proliferation. Inhibition of the c‐abl kinase with imatinib mesylate prevents increased proliferation after TGF‐β addition without affecting PAK2. These in vitro findings were extended to rats with unilateral obstructive nephropathy, a dis‐ ease model of TGF‐β‐driven renal fibrogenesis. In obstructed kidneys, PAK2 and c‐abl activity were in‐ creased but only c‐abl activation was blocked by ima‐ tinib. Treatment with imatinib did not prevent renal interstitial infiltration of macrophages or phosphoryla‐ tion and nuclear translocation of smad2/3 in ob‐ structed kidneys. In contrast, imatinib substantially inhibited an increase in the number of interstitial fibroblasts and myofibroblasts and reduced the expres‐ sion and interstitial accumulation of collagen type III, collagen type IV and fibronectin. These findings indi‐ cate that TGF‐β‐induced activation of the nonreceptor c‐abl tyrosine kinase regulates fibroblast proliferation and, by this means, is a costimulatory signal in TGF‐β‐ dependent renal fibrogenesis. Inhibition of c‐abl activ‐ ity with imatinib mesylate ameliorates experimental renal fibrosis in rats.—Wang, S., Wilkes, M. C., Leof, L. B., Hirschberg, R. Imatinib mesylate blocks a non‐ Smad TGF‐β pathway and reduces renal fibrogenesis in vivo. FASEB J. 19, 1‐11 (2005)

Proteins in the transforming growth factor-β (TGF-β) family recognize transmembrane serine/threonine kinases known as type I and type II receptors. Binding of TGF-β to receptors results in receptor down-regulation and signaling. Whereas previous work has focused on activities controlling TGF-β signaling, more recent studies have begun to address the trafficking properties of TGF-β receptors. In this report, it is shown that receptors undergo recycling both in the presence and absence of ligand activation, with the rates of internalization and recycling being unaffected by ligand binding. Recycling occurs as receptors are most likely internalized through clathrin-coated pits, and then returned to the plasma membrane via a rab11-dependent, rab4-independent mechanism. Together, the results suggest a mechanism wherein activated TGF-β receptors are directed to a distinct endocytic pathway for down-regulation and clathrin-dependent degradation after one or more rounds of recycling.

Epithelial cells in vivo form tight cell-cell associations that spatially separate distinct apical and basolateral domains. These domains provide discrete cellular processes essential for proper tissue and organ development. Using confocal imaging and selective plasma membrane domain activation, the type I and type II transforming growth factor-β (TGFβ) receptors were found to be localized specifically at the basolateral surfaces of polarized Madin-Darby canine kidney (MDCK) cells. Receptors concentrated predominantly at the lateral sites of cell-cell contact, adjacent to the gap junctional complex. Cytoplasmic domain truncations for each receptor resulted in the loss of specific lateral domain targeting and dispersion to both the apical and basal domains. Whereas receptors concentrate basolaterally in regions of direct cell-cell contact in nonpolarized MDCK cell monolayers, receptor staining was absent from areas of noncell contact. In contrast to the defined basolateral polarity observed for the TGFβ receptor complex, TGFβ ligand secretion was found to be from the apical surfaces. Confocal imaging of MDCK cells with an antibody to TGFβ1 confirmed a predominant apical localization, with a stark absence at the basal membrane. These findings indicate that cell adhesion regulates the localization of TGFβ receptors in polarized epithelial cultures and that the response to TGFβ is dependent upon the spatial distribution and secretion of TGFβ receptors and ligand, respectively.

Transforming growth factor-β (TGF-β) superfamily members regulate a wide range of biological processes by binding to two transmembrane serine/threonine kinase receptors, type I and type II. We have previously shown that the internalization of these receptors is inhibited by K+depletion, cytosol acidification, or hypertonic medium, suggesting the involvement of clathrin-coated pits. However, the involvement of the clathrin-associated adaptor complex AP2 and the identity of the AP2 subunit that binds the receptors were not known. Herein, we have studied these issues by combining studies on intact cells with in vitro assays. Using fluorescence photobleaching recovery to measure the lateral mobility of the receptors on live cells (untreated or treated to alter their coated pit structure), we demonstrated that their mobility is restricted by interactions with coated pits. These interactions were transient and mediated through the receptors' cytoplasmic tails. To measure direct binding of the receptors to specific AP2 subunits, we used yeast two-hybrid screens and in vitro biochemical assays. In contrast to most other plasma membrane receptors that bind to AP2 via the μ2 subunit, AP2/TGF-β receptor binding was mediated by a direct interaction between the β2-adaptin N-terminal trunk domain and the cytoplasmic tails of the receptors; no binding was observed to the μ2, α, or ς2 subunits of AP2 or to μ1 of AP1. The data uniquely demonstrate both in vivo and in vitro the ability of β2-adaptin to directly couple TGF-β receptors to AP2 and to clathrin-coated pits, providing the first in vivo evidence for interactions of a transmembrane receptor with β2-adaptin.