TI - Nab2 abrogates TGF-beta -induced stimulation of Type I collagen synthesis and myofibroblast differentiation . AB - We had suggested previously that endogenous Egr-1 plays an important role in mediating TGF-beta responses [10] , prompting us to consider therefore whether Nab2 might function as a negative regulator of TGF-beta signaling . To investigate the modulation of TGF-beta -induced transcriptional activation , fibroblasts were transfected with Nab2 , and incubated in the presence and absence of TGF-beta . The results showed that ectopic Nab2 abrogated TGF-beta -induced stimulation of collagen synthesis in mouse ( Fig 2A ) and human fibroblasts ( Fig 2B ) . In addition , stimulation of Egr-1 -dependent transcription (pEBS4-luc) by TGF-beta was also abrogated ( data not shown ) . Moreover , ectopic Nab2 also abrogated TGF-beta -induced stimulation of Type I collagen synthesis , COL1A2 promoter activity and COL1A1and COL1A2 mRNA expression in human skin and lung fibroblasts ( Figs 2C and D ) . Myofibroblasts play a central role in the development and progression of tissue fibrosis . Because TGF-beta stimulates myofibroblasts differentiation , and as myofibroblasts accumulation was found to be attenuated in Egr-1 null mice [26] . we investigated the regulation of myofibroblast differentiation by Nab2 . For this purpose , fibroblasts were transfected with Nab2 , and following incubation with TGF-beta for 48 h , examined by immunofluorescence . The results showed that whereas TGF-beta induced a marked increase in alpha-SMA expression and stress fiber incorporation in transfected fibroblasts , ectopic Nab2 abolished this stimulatory response ( Fig 2E ) . Induction of Smad2 PHOSphorylation , a specific marker for canonical TGF-beta signaling , was unaffected by ectopic Nab2 ( data not shown ) . Together , these results demonstrate that Nab2 has potent inhibitory effect on TGF-beta-driven stimulation of fibrotic responses independent of canonical Smad signaling . We speculated that these inhibitory effects of Nab2 involved repression of TGF-beta -induced Egr-1 signaling . To explore this possibility , additional transient transfection assays were performed . The results showed that ectopic expression of Nab2 blocked the stimulation of COL1A2-luc activity and Type I collagen synthesis induced by Egr-1 ( Figs 3A and B , and data not shown ) , whereas stimulation induced by the Nab2-resistant mutant Egr-1 could not be prevented by Nab2 . Taken together , these results not only confirm that the inhibitory effect of Nab2 was selective for Egr-1 -dependent transcriptional responses , but also serve to further underline the essential role of endogenous Egr-1 in mediating the stimulation of collagen gene expression and myofibroblasts differentiation induced by TGF-beta . To investigate the role that endogenous Nab2 might play in the physiologic regulation of collagen gene expression , two complementary approaches were taken . First , mouse embryonic fibroblasts ( MEFs ) were generated from Nab2-null mice and studied in vitro . Whereas Nab2-/- MEFs in vitro displayed morphology and proliferation rates indistinguishable from wildtype MEFs under standard culture conditions ( data not shown ) , marked alterations in their biosynthetic phenotypes were evident . Western analysis and real-time qPCR revealed substantial increases in the constitutive rate of collagen synthesis , and in COL1A1 and COL1A2 mRNA expression in Nab2-/- MEFs compared to wildtype MEFs cultured in parallel ( Figs 4A and B ) . Furthermore , incubation of Nab2-/- MEFs with TGF-beta resulted in further enhancement of collagen gene expression . A potential mechanistic explanation to account for the enhanced collagen gene transcription seen in Nab2-/- MEFs was provided by the finding that the basal activity of the Egr-1-responsive reporter pEBS4-luc was constitutively elevated in Nab2-/- MEFs , and could be partially normalized by rescuing these cells with ectopic Nab2 (Fig 4C) . These results suggest the basal Egr-1 activity is unopposed in Nab2-/- MEFs . Next , we used an siRNA knockdown approach to reduce cellular Nab2 expression . Transfection of normal dermal fibroblasts with a Nab2 siRNA reduced the baseline levels of Nab2 , and abrogated the stimulation of Nab2 expression by TGF-beta (Fig 4D) . Transfection of an irrelevant negative control siRNA ( lanes 1,2 ) had no effect on Nab2 expression . The inhibitory effect of the siRNA was specific for Nab2 , since levels of tubulin were unaffected . Down-regulation of cellular Nab2 was accompanied by significantly enhanced stimulation of collagen gene expression in response to TGF-beta ( Fig 4D , right panel ) . Together , the results of these complementary genetic and siRNA -mediated loss-of-function experiments point to an important physiologic function of cellular Nab2 in negative modulation of basal and TGF-beta -induced collagen gene expression .