Transforming growth factor-␤ (TGF-␤

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Smad6 is an inhibitory Smad that is induced by bone morphogenetic proteins (BMPs) and interferes with BMP signaling. We have isolated the mouse Smad6 promoter and identified the regions responsible for transcriptional activation by BMPs. The proximal BMP-responsive element (PBE) in the Smad6 promoter is important for the transcriptional activation by BMPs and contains a 28-base pair GC-rich sequence including four overlapping copies of the GCCGnCGC-like motif, which is a binding site for Drosophila Mad and Medea. We generated a luciferase reporter construct (3GC2-Lux) containing three repeats of the GC-rich sequence derived from the PBE. BMPs and BMP receptors induced transcriptional activation of 3GC2-Lux in various cell types, and this activation was enhanced by cotransfection of BMP-responsive Smads, i.e. Smad1 or Smad5. Moreover, direct DNA binding of BMP-responsive Smads and common-partner Smad4 to the GC-rich sequence of PBE was observed. These results indicate that the expression of Smad6 is regulated by the effects of BMP-activated Smad1/5 on the Smad6 promoter.

Objective. Members of the transforming growth factor ␤ (TGF␤) cytokine superfamily play critical roles in both homeostasis and disease. In light of their profibrotic effects, these molecules are implicated in the pathogenesis of fibrosis. In fibroblasts, TGF␤ signals through the activin receptor-like kinase 5 (ALK-5) type I TGF␤ and triggers Smad and MAP kinase signaling pathways. Because targeting of TGF␤ signaling represents a potential approach to the treatment of systemic sclerosis (SSc) and other fibrotic disorders, we investigated the modulation of intracellular TGF␤ signal transduction by SB431542, the first small-molecule inhibitor of ALK-5 to be described.Methods. Ligand-induced activation of the Smad signaling pathway in human dermal fibroblasts was examined by Western blot analysis and confocal immunocytochemistry. Modulation of profibrotic gene expression was investigated using Northern blot analysis, transient transfection assays, and confocal microscopy. Induction of TGF␤ production was evaluated by enzyme-linked immunosorbent assay.Results. SB431542 abrogated TGF␤-induced phosphorylation and nuclear importation of endogenous Smad2/3 and Smad4, and inhibited Smad3-and Systemic sclerosis (SSc) is a chronic disease of unknown etiology characterized by autoimmunity, vascular damage, and progressive fibrosis of the skin and internal organs. The pathogenesis of fibrosis is not well understood, and there are no effective treatments. Fibroblasts from lesional tissues show evidence of activation, with increased synthesis of collagen, fibronectin, tissue inhibitor of metalloproteinases 1 (TIMP-1), and plasminogen activator inhibitor 1 (PAI-1); secretion of profibrotic cytokines such as transforming growth factor Supported by grants from the NIH (AR-42309) and the Scleroderma Foundation.

Fibrosis, the hallmark of scleroderma, is characterized by excessive synthesis of collagen and extracellular matrix proteins and accumulation of myofibroblasts. Transforming growth factor-beta (TGF-beta), a potent inducer of collagen synthesis, cytokine production, and myofibroblast transdifferentiation, is implicated in fibrosis. Profibrotic TGF-beta responses are induced primarily via the type I activin-like receptor kinase 5 (ALK5) TGF-beta receptor coupled to Smad signal transducers. Here, we investigated the effect of blocking ALK5 function with SM305, a novel small-molecule kinase inhibitor, on fibrotic TGF-beta responses. In normal dermal fibroblasts, SM305 abrogated the ligand-induced phosphorylation, nuclear import, and DNA-binding activity of Smad2/3 and Smad4, and inhibited Smad2/3-dependent transcriptional responses. Furthermore, SM305 blocked TGF-beta-induced extracellular matrix gene expression, cytokine production, and myofibroblast transdifferentiation. In unstimulated scleroderma fibroblasts, SM305 caused a variable and modest reduction in type I collagen levels, and failed to abrogate constitutive nuclear accumulation of Smad2/3, or alter the proportion of smooth muscle actin stress fiber-positive fibroblasts. In vivo, SM305 prevented TGF-beta-induced Smad2/3 phosphorylation type I collagen (COL1)A2 promoter activation in dermal fibroblasts. Taken together, these results indicate that SM305 inhibits intracellular TGF-beta signaling through selective interference with ALK5-mediated Smad activation, resulting in marked suppression of profibrotic responses induced by TGF-beta in vivo and in vitro.

The non-receptor protein tyrosine kinase c-Abl regulates cell proliferation and survival. Recent studies provide evidence that implicate c-Abl as a mediator for fibrotic responses induced by Transforming growth factor-ß (TGF-ß), but the precise mechanisms underlying this novel oncogene function are unknown. Here we report that when expressed in normal fibroblasts, a constitutively active mutant of Abl mutant that causes chronic myelogenous leukemia stimulated the expression and transcriptional activity of the early growth response factor Egr-1. Mouse embryonic fibroblasts lacking c-Abl were resistant to TGF-ß. Sensitivity of these cells to TGF-ß could be rescued by wildtype c-Abl, but not by a kinase-deficient mutant form of c-Abl. Furthermore, Abl kinase activity was necessary for the induction of Egr-1 by TGF-ß in normal fibroblasts, and Egr-1 was required for stimulation of collagen by Bcr-Abl. Lesional skin fibroblasts in mice with bleomycin-induced scleroderma displayed evidence of c-Abl activation in situ, and elevated phospho-c-Abl correlated with increased local expression of Egr-1. Collectively, these results position Egr-1 downstream of c-Abl in the fibrotic response, delineate a novel Egr-1-dependent intracellular signaling mechanism that underlies the involvement of c-Abl in TGF-ß responses, and identify Egr-1 as a target of inhibition by imatinib. Furthermore, the findings demonstrate in situ activation of c-Abl paralleling the up-regulation tissue expression of Egr-1 in fibrosis. Pharmacological targeting of c-Abl and its downstream effector pathways may therefore represent a novel therapeutic approach to blocking TGF-ß-dependent fibrotic processes.

Study Design:Retrospective cohort study.Objective:The S2-alar-iliac (S2AI) technique has been described as an alternative method for pelvic fixation in place of iliac screws (ISs) in spinal deformity surgery. The objective of this study was to analyze the impact of S2AI screws on radiographical outcomes, including spinopelvic parameters.Methods:A retrospective review of 17 patients receiving ISs and 46 patients receiving S2AI screws for correction of adult spinal deformity between 2010 and 2015 with minimum 1-year follow-up was conducted. Patient data on postoperative complications, including reoperation rates and proximal junctional kyphosis (PJK), and radiographical parameters was collected and statistically analyzed.Results:With mean follow-up of 21.1 months, the overall reoperation rate was significantly lower in the S2AI group than in the IS group (21.7% vs 58.8%, P = .01), but the incidence of PJK was similar (32.6% vs 35.3%, P > .99). Moreover, the time to reoperation in the IS group was significantly shorter than in the S2AI group (P = .001), and the S2AI group trended toward a longer time to reoperation due to PJK (P = .08). There was a significantly higher change in pelvic incidence (PI) in the S2AI group (−6.0°) compared with the IS group (P = .001).Conclusions:Compared with the IS technique, the S2AI technique demonstrated a lower rate of overall reoperation, a similar rate of PJK, longer time to reoperation, and possible reduction in PI. Future studies may be warranted to clarify the mechanism of these results and how they can be translated into improved patient care.