Inhibition of Transforming Growth Factor (TGF)- 1–Induced Extracellular Matrix with a Novel Inhibitor of the TGF- Type I Receptor Kinase Activity: SB-431542

Transforming growth factor 1 (TGF1) is a potent fibrotic factor responsible for the synthesis of extracellular matrix. TGF1 acts through the TGFtype I and type II receptors to activate intracellular mediators, such as Smad proteins, the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase pathway. We expressed the kinase domain of the TGFtype I receptor [activin receptor-like kinase (ALK)5] and the substrate, Smad3, and determined that SB431542 is a selective inhibitor of Smad3 phosphorylation with an IC50 of 94 nM. It inhibited TGF1–induced nuclear Smad3 localization. The p38 mitogen-activated protein kinase inhibitors SB-203580 and SB-202190 also inhibit phosphorylation of Smad3 by ALK5 with IC50 values of 6 and 3 M, respectively. This suggests that these p38 MAPK inhibitors must be used at concentrations of less than 10 M to selectively address p38 MAPK mechanisms. However, the p38 MAPK inhibitor SB242235 did not inhibit ALK5. To evaluate the relative contribution of Smad signaling and p38 MAPK signaling in TGF1– induced matrix production, the effect of SB-431542 was compared with that of SB-242235 in renal epithelial carcinoma A498 cells. All compounds inhibited TGF1–induced fibronectin (FN) mRNA, indicating that FN synthesis is mediated in part via the p38 MAPK pathway. In contrast, SB-431542, but not the selective p38 MAPK inhibitor SB-242235, inhibited TGF1– induced collagen I 1 (col I 1). These data indicate that some matrix markers that are stimulated by TGF1 are mediated via the p38 MAPK pathway (i.e., FN), whereas others seem to be activated via ALK5 signaling independent of the p38 MAPK pathway (i.e., col I 1). Numerous reports describe TGF1 as a potent regulator of extracellular matrix. Thus, cells can be induced to produce extracellular matrix protein and inhibit protease activity by exogenous TGF1 (Nakamura et al., 1992; Ziyadeh et al., 1994). Transgenic mice that overexpress TGF1 develop severe glomerulosclerosis and liver fibrosis (Kopp et al., 1996; Kanzler et al., 1999). Finally, neutralizing antibodies against TGF1 can prevent the accumulation of extracellular matrix in models of renal disease and lung fibrosis (Border et al., 1990; Sharma et al., 1996; Ziyadeh et al., 2000). Several signaling pathways have been implicated in mediating TGF–induced extracellular matrix production and fibrosis. Transforming growth factorsignals through two highly conserved single transmembrane receptors with intracellular serine/threonine kinase domains. Specifically, the type II receptor phosphorylates threonine residues in the glycine-serine (GS)-rich domain of the ligand-occupied type I receptor (ALK5), which results in the activation of ALK5 (Wrana, 1998). ALK5, in turn, phosphorylates Smad proteins (Abdollah et al., 1997), which mediate intracellular signaling to the nucleus. TGFalso activates the p38 mitogen-activated protein kinase (MAPK) through the TGFactivated kinase, TAK1 (Hanafusa et al., 1999). Thus, the TGFreceptors can regulate gene transcription directly by activating the Smad transcription factors, indirectly via p38 MAPK, or by a combination of both pathways. Other kinases, such as JNK1 and MKK4, also seem to be involved in TGFregulation of fibronectin synthesis (Hocevar et al., 1999). Furthermore, TGF1 can activate the extracellular signal-regulated kinase through a protein synthesis ABBREVIATIONS: TGF1, transforming growth factor 1; GS, glycine-serine; ALK, activin receptor-like kinase; MAPK, mitogen-activated protein kinase; SB-203580, 4-[4 -fluorophenyl]-2-[4 -methylsulfinylphenyl]-5-[4 -pyridyl] imidazole; SB-431542, 4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl1H-imidazol-2-yl)-benzamide; SB-242235, 4-[4-(4-flurophenyl)-1-(4-piperidinyl)-1H-imidazol-5-yl]-2-methoxypyrimidine; GST, glutathione S-transferase; RPTEC, renal proximal tubule epithelial cells; XTT, 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxyaniline; BMP, bone morphogenetic protein; PBS, phosphate-buffered saline; PAI-1, plasminogen activator inhibitor-1; TSP-1, thrombospondin-1; FN, fibronectin; rpL32, ribosomal protein L32; RT-PCR, reverse transcription-polymerase chain reaction; SB-202190, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole. 0026-895X/02/6201-58–64$7.00 MOLECULAR PHARMACOLOGY Vol. 62, No. 1 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1514/989396 Mol Pharmacol 62:58–64, 2002 Printed in U.S.A. 58 at A PE T Jornals on A uust 8, 2017 m oharm . aspeurnals.org D ow nladed from requiring a mechanism that may involve basic fibroblast growth factor (Finlay et al., 2000). It is clear that TGFcan interact with several kinase pathways to influence many components of the extracellular matrix. Although it has been shown that the p38 MAPK inhibitor SB-203580 can inhibit ALK5 autophosphorylation with an IC50 of 20 M (Eyers et al., 1998), more selective inhibitors have not been applied to TGFregulation of extracellular matrix. We characterized several compounds for their ability to selectively inhibit ALK5 or p38 MAPK to delineate the relative contribution of Smad and p38 MAPK activation in TGFregulation of extracellular matrix. In this study, therefore, we examine the effects of the selective ALK5 inhibitor SB-431542 (Callahan et al., 2002) and the selective p38 MAPK inhibitor SB-242235 (Badger et al., 2000) on TGF1–induced fibronectin, collagen I 1, thrombospondin-1, and plasminogen activator inhibitor-1 mRNA. Materials and Methods Expression of Recombinant Protein. The kinase domain of ALK5, amino acids 200 to the C terminus, which lacks the GS region, and the full-length Smad3 protein were expressed as N-terminal glutathione S-transferase (GST) fusion proteins in baculovirus expression system. Proteins were purified with glutathione Sepharose beads 4B (Amersham Biosciences, Uppsala, Sweden) (Roshak et al.,