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Interleukin-7 and Transforming Growth Factor-β Play Counter-regulatory Roles in Protein Kinase C-δ-dependent Control of Fibroblast Collagen Synthesis in Pulmonary Fibrosis

2004; Elsevier BV; Volume: 279; Issue: 27 Linguagem: Inglês

10.1074/jbc.c400115200

1083-351X

Ling Zhang, Michael P. Keane, Li Zhu, Sherven Sharma, Enrique Rozengurt, Robert M. Strieter, Steven M. Dubinett, Min Huang,

Neonatal Respiratory Health Research

Transforming growth factor-β (TGF-β) is a potent fibrogenic factor responsible for promoting synthesis of extracellular matrix. Interleukin-7 (IL-7) inhibits TGF-β signaling by up-regulating Smad7, a major inhibitor of the Smad family. In a variety of cells, TGF-β-mediated activation of target genes requires active protein kinase C-δ (PKC-δ) in addition to Smads (1Kucich U. Rosenbloom J.C. Abrams W.R. Rosenbloom J. Am. J. Respir. Cell Mol. Biol. 2002; 26: 183-188Crossref PubMed Scopus (104) Google Scholar). We determined the role of PKC-δ in the regulation of pulmonary fibroblast collagen synthesis in response to TGF-β and IL-7 stimulation. Here we show that TGF-β and IL-7 have opposing effects on PKC-δ; TGF-β stimulates, while IL-7 inhibits, PKC-δ activity. IL-7 inhibits TGF-β-induced PKC-δ phosphorylation at Ser-645 and Thr-505. Inhibition of PKC-δ with specific small inhibitory RNA restores TGF-β-mediated induction of Smad7 and in parallel significantly reduces TGF-β-mediated collagen synthesis. Thus, PKC-δ may play a critical role in the pathogenesis of pulmonary fibrosis and may serve as a molecular target for therapeutic intervention to suppress fibrosis. Transforming growth factor-β (TGF-β) is a potent fibrogenic factor responsible for promoting synthesis of extracellular matrix. Interleukin-7 (IL-7) inhibits TGF-β signaling by up-regulating Smad7, a major inhibitor of the Smad family. In a variety of cells, TGF-β-mediated activation of target genes requires active protein kinase C-δ (PKC-δ) in addition to Smads (1Kucich U. Rosenbloom J.C. Abrams W.R. Rosenbloom J. Am. J. Respir. Cell Mol. Biol. 2002; 26: 183-188Crossref PubMed Scopus (104) Google Scholar). We determined the role of PKC-δ in the regulation of pulmonary fibroblast collagen synthesis in response to TGF-β and IL-7 stimulation. Here we show that TGF-β and IL-7 have opposing effects on PKC-δ; TGF-β stimulates, while IL-7 inhibits, PKC-δ activity. IL-7 inhibits TGF-β-induced PKC-δ phosphorylation at Ser-645 and Thr-505. Inhibition of PKC-δ with specific small inhibitory RNA restores TGF-β-mediated induction of Smad7 and in parallel significantly reduces TGF-β-mediated collagen synthesis. Thus, PKC-δ may play a critical role in the pathogenesis of pulmonary fibrosis and may serve as a molecular target for therapeutic intervention to suppress fibrosis. Pulmonary fibrosis is characterized by disturbances of extracellular matrix protein deposition resulting from fibroblast activation and proliferation (2Broekelmann T.J. Limper A.H. Colby T.V. McDonald J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6642-6646Crossref PubMed Scopus (688) Google Scholar). TGF-β 1The abbreviations used are: TGF-β, transforming growth factor-β; PKC-δ, protein kinase C-δ; IL-7, interleukin-7; IPF, idiopathic pulmonary fibrosis; PFF, pulmonary fibrosis fibroblasts; NF, normal fibroblasts; siRNA, small inhibitory RNA; RT, reverse transcriptase. is a major driving force in the pathogenesis of pulmonary fibrosis (2Broekelmann T.J. Limper A.H. Colby T.V. McDonald J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6642-6646Crossref PubMed Scopus (688) Google Scholar). Overexpression of TGF-β1 in pulmonary fibrosis contributes to the dysregulation of normal homeostatic mechanisms at multiple levels; it enhances synthesis and deposition of extracellular matrix components including collagen and can also alter the balance of matrix metalloproteinases and their inhibitors (2Broekelmann T.J. Limper A.H. Colby T.V. McDonald J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6642-6646Crossref PubMed Scopus (688) Google Scholar, 3Khalil N. Bereznay O. Sporn M. Greenberg A.H. J. Exp. Med. 1989; 170: 727-737Crossref PubMed Google Scholar). TGF-β signaling and regulation are mediated by a family of Smad proteins (4Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3999) Google Scholar). TGF-β induces phosphorylation of Smad2 and Smad3, which form hetero-oligomeric complex with Smad4 that translocate to the nucleus, where they recognize Smad binding elements to regulate transcriptional responses of target genes (4Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3999) Google Scholar). Smad7 is a major inhibitory member of the Smad family (4Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3999) Google Scholar). Smad7 inhibits TGF-β-mediated phosphorylation of Smad2/3 and competes with Smad2/3 for the binding to TGF-β receptor. TGF-β rapidly induces expression of Smad7 mRNA, suggesting a negative feedback loop to control TGF-β signaling (4Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3999) Google Scholar). We have demonstrated that IL-7 possesses potent antifibrotic activities in vitro and in vivo (5Huang M. Sharma S. Zhu L.X. Keane M.P. Luo J. Zhang L. Burdick M.D. Lin Y.Q. Dohadwala M. Gardner B. Batra R.K. Strieter R.M. Dubinett S.M. J. Clin. Invest. 2002; 109: 931-937Crossref PubMed Google Scholar). IL-7 inhibits TGF-β signaling through the induction of Smad7 (5Huang M. Sharma S. Zhu L.X. Keane M.P. Luo J. Zhang L. Burdick M.D. Lin Y.Q. Dohadwala M. Gardner B. Batra R.K. Strieter R.M. Dubinett S.M. J. Clin. Invest. 2002; 109: 931-937Crossref PubMed Google Scholar). In addition to Smads, TGF-β requires active PKC-δ to activate its target genes in a variety of cells including pulmonary fibroblasts (1Kucich U. Rosenbloom J.C. Abrams W.R. Rosenbloom J. Am. J. Respir. Cell Mol. Biol. 2002; 26: 183-188Crossref PubMed Scopus (104) Google Scholar). PKC-δ, a ubiquitously expressed kinase involved in a variety of cellular signaling pathways, has been implicated in de novo synthesis of extracellular matrix components (6Runyan C.E. Schnaper H.W. Poncelet A.C. Am. J. Physiol. 2003; 285: F413-F422Crossref PubMed Scopus (93) Google Scholar). PKC-δ participates in the up-regulation of type I (COL1A1) and type III (COL3A1) collagen gene expression mediated by TGF-β in scleroderma fibroblasts (7Jimenez S.A. Gaidarova S. Saitta B. Sandorfi N. Herrich D.J. Rosenbloom J.C. Kucich U. Abrams W.R. Rosenbloom J. J. Clin. Invest. 2001; 108: 1395-1403Crossref PubMed Scopus (77) Google Scholar). Based on the importance of PKC-δ in TGF-β signaling, we determined whether PKC-δ modulated collagen synthesis in response to TGF-β and IL-7 stimulation in pulmonary fibroblasts. Cell Culture—Isolation, passage, and specific cell marker characterization of human pulmonary fibroblasts were performed as described previously (5Huang M. Sharma S. Zhu L.X. Keane M.P. Luo J. Zhang L. Burdick M.D. Lin Y.Q. Dohadwala M. Gardner B. Batra R.K. Strieter R.M. Dubinett S.M. J. Clin. Invest. 2002; 109: 931-937Crossref PubMed Google Scholar). Fibroblasts obtained from patients with idiopathic pulmonary fibrosis (PFF) or non-fibrotic lung diseases (NF) were maintained in 5% CO2 in air as monolayers at 37 °C in 75-cm2 tissue culture flasks containing 20 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mm glutamine (JRH Biosciences, Lenexa, KS). Cytokines and Antibodies—Human recombinant TGF-β1 (3.2 × 104 units/μg) and recombinant human IL-7 (105 units/μg) were obtained from R & D Systems, Minneapolis, MN. Rabbit anti-PKC-δ, rabbit anti-PKC-δ-[Ser645], and rabbit anti-PKC-δ-[pTyr311] antibodies were obtained from BIOSOURCE International, Inc., Camarillo, CA. Rabbit anti-PKC-δ [pThr505] antibody was obtained from Cell Signaling Technology, Inc., Beverly, MA. Lyophilized human recombinant TGF-β1 was reconstructed in sterile 4 mm HCl containing 0.1% bovine serum albumin in a final concentration of 10 μg/ml and stored at –70 °C in aliquots. Northern Blot Analysis—High quality total RNA from 107 cells of NF and PFF was prepared by the TRIzol method (Invitrogen). Northern blotting was performed as previously described (8Huang M. Lindahl R. Arch. Biochem. Biophys. 1990; 277: 296-300Crossref PubMed Scopus (17) Google Scholar). We used the following unique 30-mer oligonucleotide probes for the detection of human COL1A1 and COL3A1 gene expression: 5′-CAGTTCTTGGTCTCGTCACAGATCACGTCA-3′ (human COL1A1 probe, corresponding to the coding region 332–361) and 5′-TTCCATTTTCTCCTGGAGGACCACCTGTAC-3′ (human COL3A1 probe, corresponding to the coding region 2051–2080). Duplicate filters were hybridized with 32P-radiolabeled human β-actin cDNA probe serving as controls for equal loading and transfer. The filters were exposed to an x-ray film for documentation and analyzed using phosphor imaging equipment (Molecular Imager, Bio-Rad). PKC-δ Activity Assays—PFF and NF were incubated in medium alone, in medium containing IL-7 (100 ng/ml), in medium containing TGF-β1 (10 ng/ml), or in medium containing IL-7 and TGF-β1 for 1 h at 37 °C. 5 × 106 Cells of each sample were lysed in phosphorylation lysis buffer containing 150 mm NaCl, 200 μm sodium orthovanadate, 10 mm sodium prophosphate, 100 mm sodium fluoride, 1.5 mm MgCl2, 10% glycerol, 1 mm EDTA, 0.5% Triton X-100, and proteinase inhibitors. Cell lysates were immunoprecipitated with a specific anti-PKC-δ antibody (BIOSOURCE International, Inc.). The immunoprecipitates were washed three times with phosphorylation lysis buffer and two times with kinase buffer, which contains 25 mm Tris-HCl (pH 7.4), 5 mm MgCl2, 0.5 mm EDTA, 1 mm dithiothreitol, 20 μg of phosphatidylserine, and 20 mm ATP. The immunoprecipitates were resuspended in 30 μl of kinase buffer containing 5 μg of an exogenous substrate histone H1 and 20 μCi of [γ-32P]ATP and incubated at 25 °C for 30 min. The reaction was terminated by the addition of an equal volume of SDS sample buffer. The PKC-δ activity was determined by the amount of 32P-histone H1 produced as detected by autoradiography following SDS-PAGE electrophoresis. Western Blot Analysis—PFF and NF under standard growth conditions or in medium supplemented with cytokines were suspended in 1× phosphate-buffered saline buffer containing 1 mm MgCl2, protease inhibitors (10 μl/ml), and 0.1% Triton X-100. The cell suspension was sonicated and then centrifuged at 11,000 × g for 10 min at 4 °C. Protein content of each sample was determined using the protein assay kit (Bio-Rad). Aliquots containing 80 μg of lysate were boiled for 5 min and electrophoresed on 7.5% polyacrylamide gels. The gels were transferred to Immuno-Blot membranes (Bio-Rad), blocked for 1 h with 5% nonfat milk, and then incubated with specific primary antibodies. The immunoreactive proteins were detected using a ProtoBlot II AP System kit (Promega Corp., Madison, WI). PKC-δ siRNA—PKC-δ siRNA Duplex1 for the target sequence 5′-AAG ATG AAG GAG GCG CTC AG-3′ was obtained from Qiagen (Valencia, CA). Two micrograms of PKC-δ siRNA were premixed with TransMessenger Transfection Reagent (Qiagen Inc., Valencia, CA) in a ratio of 1:2 to 1:8. PFF and NF were then transfected with PKC-δ siRNA mixture or solvent only as controls for 3 h under standard growth condition. Real-time RT-PCR—Five hundred nanograms of total RNA from PFF (n = 2) and NF (n = 3) were used for the real-time RT-PCR analysis using unique target gene primers or housekeeping gene β-actin primers serving as controls for equal loading. Primer pairs for the optimal real-time RT-PCR results were designed with the assistance of ongoing consultation with the technical team of the manufacturer (Bio-Rad). We used the following unique primer pairs for the detection of human Smad7 mRNA by the real-time RT-PCR: 5′-CTGCAACCCCCATCACCTTA-3′ (Smad7 sense primer) and 5′-CCCTGTTTCAGCGGAGGAA-3′ (Smad7 antisense primer). We used the following unique primer pairs for the detection of human β-actin mRNA serving as controls for equal loading by the real-time RT-PCR: 5′-GATGAGATTGGCATGGCTTT-3′ (β-actin sense primer) and 5′-CACCTTCACCGTTCCAGTTT-3′ (β-actin antisence primer). The Smad7 primer pairs correspond to the coding region 576–709 and the amplified products are 134 bp in length. The β-actin primer pairs correspond to the coding region 1233–1332, and the amplified products are 100 bp in length. Both Smad7 and β-actin amplified products have minimal secondary structures, and the melt curve analysis showed no nonspecific amplicon or primer/dimer formation. A QuantiTect CYBRGreen RT-PCR kit (Qiagen Inc.) was used to prepare the real-time RT-PCR reaction mixture. Real-time RT-PCR was performed at 50 °C for 1 h using unique antisense primers for the RT step followed by 45 cycles of amplification using an iCycler iQ Real-Time PCR Detection System (Bio-Rad). The real-time RT-PCR data were simultaneously analyzed using the same system. Collagen Assays—Collagen assay was performed using the Sircol collagen assay method (Accurate Chemical & Scientific Corp., Westbury, NY) as described previously (5Huang M. Sharma S. Zhu L.X. Keane M.P. Luo J. Zhang L. Burdick M.D. Lin Y.Q. Dohadwala M. Gardner B. Batra R.K. Strieter R.M. Dubinett S.M. J. Clin. Invest. 2002; 109: 931-937Crossref PubMed Google Scholar). Statistical Analysis—The results are presented as means ± S.D. from single experiment that are representative of at least three replicate experiments. Significance between experimental versus control values was calculated using the Student's t test. IL-7 Inhibits TGF-β-induced COL1A1 and COL3A1 Gene Expression in PFF—To determine whether IL-7 modulates TGF-β-induced collagen synthesis at the level of regulating COL1A1 and COL3A1 gene expresion, PFF were incubated in medium only, in medium containing acid-activated recombinant TGF-β1 (10 ng/ml), or in medium containing TGF-β1 plus IL-7 (100 ng/ml). When used at this concentration TGF-β1 does not significantly impact fibroblast proliferation at 24 h (5Huang M. Sharma S. Zhu L.X. Keane M.P. Luo J. Zhang L. Burdick M.D. Lin Y.Q. Dohadwala M. Gardner B. Batra R.K. Strieter R.M. Dubinett S.M. J. Clin. Invest. 2002; 109: 931-937Crossref PubMed Google Scholar). After a 24-h incubation, cells were harvested for Northern blot analysis using 32P-labeled oligonucleotide probes that recognize unique human COL1A1 and COL3A1 transcripts. IL-7 significantly reduced TGF-β-mediated induction of COL1A1 and COL3A1 mRNA levels (Fig. 1). IL-7 and TGF-β Have Opposing Effects on PKC-δ Activity in PFF—We previously showed that IL-7 inhibited TGF-β signaling in a Smad-7-dependent manner (5Huang M. Sharma S. Zhu L.X. Keane M.P. Luo J. Zhang L. Burdick M.D. Lin Y.Q. Dohadwala M. Gardner B. Batra R.K. Strieter R.M. Dubinett S.M. J. Clin. Invest. 2002; 109: 931-937Crossref PubMed Google Scholar). Based on the importance of PKC-δ in TGF-β signaling, we determined the role of PKC-δ in IL-7-mediated inhibition of TGF-β signaling by analysis of PKC-δ kinase activity. PFF and NF were exposed to the following conditions: medium only, IL-7 (100 ng/ml), TGF-β (10 ng/ml), or IL-7 + TGF-β at 37 °C for 1 h (1Kucich U. Rosenbloom J.C. Abrams W.R. Rosenbloom J. Am. J. Respir. Cell Mol. Biol. 2002; 26: 183-188Crossref PubMed Scopus (104) Google Scholar, 6Runyan C.E. Schnaper H.W. Poncelet A.C. Am. J. Physiol. 2003; 285: F413-F422Crossref PubMed Scopus (93) Google Scholar). The PKC-δ activity was determined by the amount of 32P-histone H1 produced as detected by autoradiography following SDS-PAGE electrophoresis. The results show that IL-7 inhibited both constitutive and TGF-β-induced PKC-δ activity in PFF but had no effect on PKC-δ activity in NF (Fig. 2). Thus, IL-7 and TGF-β have opposing effect on PKC-δ activity; IL-7 is inhibitory, while TGF-β is stimulatory, in PFF. IL-7 Inhibits TGF-β-mediated Phosphorylation of PKC-δ at Thr-505 and Ser-645 in PFF—PKC-δ mediates a wide range of biological responses, and its activity is controlled by distinct phosphorylation sites (9Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (976) Google Scholar). To determine the characteristic phosphorylation patterns of PKC-δ activation regulated by IL-7 and TGF-β in NF and PFF, we performed Western blot analysis using a panel of specific monoclonal antibodies including anti-total PKC-δ, anti-phospho-PKC-δ-[pThr505], anti-phospho-PKC-δ-[pTyr311], and anti-phospho-PKC-δ-[pSer645] antibodies. PFF and NF were incubated in the following conditions: medium only, IL-7 (100 ng/ml), TGF-β (10 ng/ml), and IL-7 + TGF-β at 37 °C for 1 h (6Runyan C.E. Schnaper H.W. Poncelet A.C. Am. J. Physiol. 2003; 285: F413-F422Crossref PubMed Scopus (93) Google Scholar). As shown in Fig. 3, TGF-β stimulated phosphorylation of PKC-δ at Ser-645 and Thr-505 in PFF, while IL-7 inhibited this process. Consistent with our previous findings, PKC-δ phosphorylation status in NF was not influenced by IL-7 and TGF-β. The results suggest that the opposing effects of IL-7 and TGF-β on PKC-δ activity in PFF may be due to the differential regulation of post-translational PKC-δ phosphorylation. Inhibition of PKC-δ with Specific siRNA Significantly Increased Smad7 mRNA Levels and, in Parallel, Reduced TGF-β-mediated Collagen Synthesis in PFF—PKC-δ is implicated in de novo synthesis of connective tissue and extracellular matrix components (9Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (976) Google Scholar). For example, TGF-β-stimulated PKC-δ activity positively regulates Smad transcriptional activity resulting in up-regulation of the COL1A2 gene expression in renal mesangial cells (6Runyan C.E. Schnaper H.W. Poncelet A.C. Am. J. Physiol. 2003; 285: F413-F422Crossref PubMed Scopus (93) Google Scholar). We have shown that IL-7 inhibits TGF-β-mediated collagen synthesis through the induction of Smad7 (5Huang M. Sharma S. Zhu L.X. Keane M.P. Luo J. Zhang L. Burdick M.D. Lin Y.Q. Dohadwala M. Gardner B. Batra R.K. Strieter R.M. Dubinett S.M. J. Clin. Invest. 2002; 109: 931-937Crossref PubMed Google Scholar). Based on the importance of PKC-δ in a variety of cellular signaling pathways including TGF-β signaling, we tested whether PKC-δ may play a role in the regulation of collagen synthesis in response to TGF-β and IL-7 stimulation in PFF. In preliminary studies, we tested the conditions for optimal gene silencing with PKC-δ siRNA in PFF. As demonstrated by Western blot analysis in Fig. 4A, PKC-δ siRNA, mixed with transfection reagent in a ratio of 1:8, provided an optimal gene silencing effect of PKC-δ (>95% reduction of gene products), while β-actin levels, serving as controls for equal loading, remained unchanged. To determine the role of PKC-δ in Smad7 gene regulation, NF (n = 2) and PFF (n = 3) were preincubated in medium with or without PKC-δ siRNA for 24 h. TGF-β1 (10 ng/ml) was then added to the cultures, and cells were incubated for additional 90 min prior to isolation of total RNA (10Patil S. Wildey G.M. Brown T.L. Choy L. Derynck R. Howe P.H. J. Biol. Chem. 2000; 275: 38363-38370Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Smad7 mRNA levels of each sample were determined by realtime RT-PCR analysis. As shown in Fig. 4B. TGF-β significantly increased Smad7 mRNA expression levels up to 16-fold in NF, while TGF-β had no significant effect on Smad7 mRNA levels in PFF (p < 0.01). However, when PFF were incubated with TGF-β + PKC-δ siRNA, Smad7 mRNA levels only increased 7.3-fold compared with cells incubated with TGF-β alone (p < 0.05). In comparison, inhibition of PKC-δ with specific siRNA in NF did not show significant differences in Smad7 mRNA levels. The results suggest that PKC-δ is important in TGF-β-mediated regulation of Smad7 gene expression in lung fibroblasts. Based on our results and those of others (6Runyan C.E. Schnaper H.W. Poncelet A.C. Am. J. Physiol. 2003; 285: F413-F422Crossref PubMed Scopus (93) Google Scholar, 9Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (976) Google Scholar), we hypothesized that TGF-β-mediated collagen synthesis in PFF could be blocked by the inhibition of PKC-δ. PFF (n = 3) was cultured in medium alone, in medium containing TGF-β1 (10 ng/ml), or in medium containing TGF-β1 plus PKC-δ siRNA for 24 h. The inhibition of PKC-δ with siRNA significantly reduced collagen levels (47–100% reduction) in response to TGF-β stimulation in PFF (Fig. 4C). Thus, PKC-δ may play a critical role in the regulation of fibroblast collagen synthesis in pulmonary fibrosis. Idiopathic pulmonary fibrosis (IPF) is a devastating disease with less than a 50% 5-year survival (11Gross T. Hunninghake M. N. Engl. J. Med. 2001; 345: 517-525Crossref PubMed Scopus (889) Google Scholar). While steroids and other immunosuppressive agents serve as the standard treatment for IPF, these agents are ineffective. The development of new therapeutic paradigms is clearly a clinical priority. The pathogenesis of pulmonary fibrosis includes the deterioration of the normal homeostatic mechanisms regulating the equilibrium between the synthesis and breakdown of the extracellular matrix (2Broekelmann T.J. Limper A.H. Colby T.V. McDonald J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6642-6646Crossref PubMed Scopus (688) Google Scholar, 3Khalil N. Bereznay O. Sporn M. Greenberg A.H. J. Exp. Med. 1989; 170: 727-737Crossref PubMed Google Scholar, 11Gross T. Hunninghake M. N. Engl. J. Med. 2001; 345: 517-525Crossref PubMed Scopus (889) Google Scholar, 12Sheppard D. J. Clin. Invest. 2001; 107: 1501-1502Crossref PubMed Scopus (70) Google Scholar). TGF-β plays a critical role in the pathogenesis of pulmonary fibrosis by enhancing synthesis and deposition of extracellular matrix components including collagen (2Broekelmann T.J. Limper A.H. Colby T.V. McDonald J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6642-6646Crossref PubMed Scopus (688) Google Scholar, 3Khalil N. Bereznay O. Sporn M. Greenberg A.H. J. Exp. Med. 1989; 170: 727-737Crossref PubMed Google Scholar). Based on the importance of TGF-β in the pathogenesis of pulmonary fibrosis, we investigated the mechanisms of dysregulation of TGF-β signaling in PFF. We reported previously that IL-7 has the capacity to block TGF-β signaling by up-regulation of Smad7, a major inhibitory member of the Smad family. In vitro, IL-7 inhibits fibroblast collagen deposition in a Smad-7-dependent manner (5Huang M. Sharma S. Zhu L.X. Keane M.P. Luo J. Zhang L. Burdick M.D. Lin Y.Q. Dohadwala M. Gardner B. Batra R.K. Strieter R.M. Dubinett S.M. J. Clin. Invest. 2002; 109: 931-937Crossref PubMed Google Scholar). In vivo, IL-7 limits pulmonary fibrosis (5Huang M. Sharma S. Zhu L.X. Keane M.P. Luo J. Zhang L. Burdick M.D. Lin Y.Q. Dohadwala M. Gardner B. Batra R.K. Strieter R.M. Dubinett S.M. J. Clin. Invest. 2002; 109: 931-937Crossref PubMed Google Scholar). Because collagen types I and III are major components of extracellular matrix in pulmonary fibrosis (2Broekelmann T.J. Limper A.H. Colby T.V. McDonald J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6642-6646Crossref PubMed Scopus (688) Google Scholar, 3Khalil N. Bereznay O. Sporn M. Greenberg A.H. J. Exp. Med. 1989; 170: 727-737Crossref PubMed Google Scholar), we first investigated the effect of IL-7 on TGF-β-mediated induction of COL1A1 and COL3A1 gene expression in PFF. Consistent with a marked reduction in collagen synthesis as previously reported, the current results show that IL-7 inhibits TGF-β-mediated induction of COL1A1 and COL3A1 gene expression in PFF. Thus, IL-7 inhibits TGF-β-mediated collagen synthesis at both mRNA and protein levels. PKC-δ is a member of the PKC family of serine-threonine kinases, which play important roles in signaling for various cytokine receptors. Specifically relevant to our current study, the Smad cascade and PKC-δ mediate TGF-β1-induced target gene expression in a variety of cell types (1Kucich U. Rosenbloom J.C. Abrams W.R. Rosenbloom J. Am. J. Respir. Cell Mol. Biol. 2002; 26: 183-188Crossref PubMed Scopus (104) Google Scholar, 6Runyan C.E. Schnaper H.W. Poncelet A.C. Am. J. Physiol. 2003; 285: F413-F422Crossref PubMed Scopus (93) Google Scholar). Previous studies indicate that inhibition of PKC-δ results in marked reduction in TGF-β-induced COL1A1 and COL3A1 mRNA expression (13Li W. Mischak H. Yu J.C. Wang L.M. Mushinski J.F. Heidaran M.A. Pierce J.H. J. Biol. Chem. 1994; 269: 2349-2352Abstract Full Text PDF PubMed Google Scholar), suggesting that PKC-δ may play an important role in the regulation of TGF-β-mediated collagen synthesis. In the current study, we have shown that TGF-β stimulates, while IL-7 inhibits, PKC-δ activity in PFF. These opposing effects of TGF-β and IL-7 on PKC-δ correlate well with our previously published work; TGF-β stimulates, while IL-7 inhibits, collagen synthesis in PFF. TGF-β stimulates phosphorylation of PKC-δ at Ser-645 and Thr-505, while IL-7 inhibits this process in PFF. TGF-β receptors have intrinsic serine-threonine kinase activity, which serves as a key component in the activation of intracellular TGF-β signaling (1Kucich U. Rosenbloom J.C. Abrams W.R. Rosenbloom J. Am. J. Respir. Cell Mol. Biol. 2002; 26: 183-188Crossref PubMed Scopus (104) Google Scholar, 9Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (976) Google Scholar). Our results suggest that IL-7 may inhibit TGF-β-mediated PKC-δ phosphorylation of Ser-645 and Thr-505 by interfering with TGF-β receptor intrinsic kinase activity in cells in which PKC-δ is a downstream target of TGF-β receptor serine-threonine kinases. In addition to phosphorylation on Ser-645 and Thr-505, PKC-δ can also be phosphorylated by various tyrosine kinases including Fyn, Src, Lyn, Lck, and Abl (1Kucich U. Rosenbloom J.C. Abrams W.R. Rosenbloom J. Am. J. Respir. Cell Mol. Biol. 2002; 26: 183-188Crossref PubMed Scopus (104) Google Scholar, 9Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (976) Google Scholar). Our results show that TGF-β and IL-7 have no effect on PKC-δ phosphorylation at Tyr-311 in both PFF and NF. Thus, Tyr-311 phosphorylation may not be required for PKC-δ activity in human pulmonary fibroblasts. We (5Huang M. Sharma S. Zhu L.X. Keane M.P. Luo J. Zhang L. Burdick M.D. Lin Y.Q. 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These differences have been demonstrated at multiple levels including TGF-β receptor expression, expression of cell surface receptors or signal transduction mediators, regulation of TGF-β signaling, and response to cytokines or other stimulators. Normal fibroblasts have the capacity to produce TGF-β in response to a variety of stimuli. This production of TGF-β then serves to control and mediate processes such as wound repair. However, following TGF-β production, regulatory mechanisms must also be initiated to limit TGF-β signaling and avoid, for example, uncontrolled collagen production and fibrogenesis. Thus, exposure of normal cells to TGF-β leads to the rapid induction of Smad7 mRNA expression. In the current study, Smad7 mRNA levels rapidly increased in response to TGF-β stimulation in NF, while no significant differences were observed in PFF. The results suggest that the normal negative feedback regulatory mechanism for TGF-β signaling may be impaired in PFF. Thus, NF are endowed with balanced fibrogenic and antifibrogenic regulatory mechanisms and appear to be resistant to aberrant signals seen in pulmonary fibrosis. In comparison, PFF have a fibrogenic phenotype. Importantly, inhibition of PKC-δ (>95% inhibition) with specific siRNA significantly increased Smad7 mRNA levels and, in parallel, reduced TGF-β-mediated collagen synthesis in PFF. The results suggest that the inhibition of PKC-δ may at least partially restore the negative feedback of TGF-β in PFF by facilitating an increase in Smad7. IL-7 is a cytokine within the IL-2 receptor common γ chain (IL-2Rγ(c) superfamily (18Xiao H. Yin T. Wang X.Y. Uchida T. Chung J. White M.F. Yang Y.C. J. Biol. Chem. 2002; 277: 8091-8098Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). IL-7 receptor (IL-7Rα/γ(c)) is comprised of a unique α chain (IL-7Rα) and a common γ (IL-7Rγ) subunit shared by other cytokine receptors including IL-2, IL-4, IL-9, and IL-15 (19Kondo M. 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In vitro, IL-7 has the capacity to inhibit fibroblast TGF-β signaling in a JAK1/STAT1-dependent manner. In vivo, IL-7 decreases bleomycin-induced pulmonary fibrosis. Here we show for the first time that the counter-regulatory roles mediated by IL-7 and TGF-β in pulmonary fibroblast Smad7 expression and collagen synthesis are PKC-δ-dependent. PKC-δ may play a critical role in the pathogenesis of pulmonary fibrosis and serve as a molecular target for therapeutic intervention. The development of pathogenesis-based therapy for pulmonary fibrosis offers an entirely new avenue for therapeutic intervention.