Scleroderma Fibroblasts Demonstrate Enhanced Activation of Akt (Protein Kinase B) In Situ
2005; Elsevier BV; Volume: 124; Issue: 2 Linguagem: Inglês
10.1111/j.0022-202x.2004.23559.x
ISSN1523-1747
AutoresJae‐Bum Jun, Melanie K. Kuechle, Jun‐Ki Min, Seung Cheol Shim, Gi‐Ok Kim, Vivianne Montenegro, Joseph H. Korn, Keith B. Elkon,
Tópico(s)Dermatologic Treatments and Research
ResumoRecent studies suggest that, in addition to activation and hypersecretion of matrix components, fibroblasts from patients with systemic sclerosis (SSc) are relatively resistant to apoptosis. Transforming growth factor-β (TGF)-β is strongly implicated in the pathogenesis of SSc and we and others have shown that TGF-β can activate Akt, a kinase with potent anti-apoptotic effects. To determine whether Akt was activated in SSc, we quantified phospho-Akt expression in skin fibroblasts in vitro by western blot analysis and a functional kinase assay. In addition, the relative proportion of fibroblasts containing activated Akt in was quantified by immunohistochemistry on skin sections insitu. Analysis of Akt phosphorylation of skin fibroblasts in vitro suggested increased phosphorylation of Akt, and evaluation of skin sections by immunohistochemistry revealed significantly higher percentages of fibroblasts that stained for phospho-Akt compared with controls (78%±14.0% vs 13%±9%, p<0.001). In addition, co-incident staining of phospho-Akt and α-smooth muscle actin was observed in some fibroblasts. These findings indicate that Akt is activated insitu in skin fibroblasts from patients with SSc. Akt activation may contribute to resistance to apoptosis, selection of disease-inducing fibroblasts, and, possibly, myofibroblasts. Recent studies suggest that, in addition to activation and hypersecretion of matrix components, fibroblasts from patients with systemic sclerosis (SSc) are relatively resistant to apoptosis. Transforming growth factor-β (TGF)-β is strongly implicated in the pathogenesis of SSc and we and others have shown that TGF-β can activate Akt, a kinase with potent anti-apoptotic effects. To determine whether Akt was activated in SSc, we quantified phospho-Akt expression in skin fibroblasts in vitro by western blot analysis and a functional kinase assay. In addition, the relative proportion of fibroblasts containing activated Akt in was quantified by immunohistochemistry on skin sections insitu. Analysis of Akt phosphorylation of skin fibroblasts in vitro suggested increased phosphorylation of Akt, and evaluation of skin sections by immunohistochemistry revealed significantly higher percentages of fibroblasts that stained for phospho-Akt compared with controls (78%±14.0% vs 13%±9%, p<0.001). In addition, co-incident staining of phospho-Akt and α-smooth muscle actin was observed in some fibroblasts. These findings indicate that Akt is activated insitu in skin fibroblasts from patients with SSc. Akt activation may contribute to resistance to apoptosis, selection of disease-inducing fibroblasts, and, possibly, myofibroblasts. glycogen synthase kinase-3 hematoxylin & eosin phosphaditylinositol 3 smooth muscle actin systemic sclerosis transforming growth factor-β Systemic sclerosis (SSc) is a chronic autoimmune, vascular, and fibrotic disease culminating in excessive accumulation of extracellular matrix and fibrosis of the skin and internal organs such as the heart, lungs, and intestinal tract (LeRoy et al., 1988LeRoy E.C. Black C. Fleischmajer R. et al.Scleroderma (systemic sclerosis): Classification, subsets and pathogenesis.J Rheumatol. 1988; 15: 202-205PubMed Google Scholar). Increased matrix synthesis by scleroderma fibroblasts is a phenotype maintained in vitro, suggesting that the pathogenetic process of SSc favors the growth and/or survival of subpopulations of pro-fibrotic fibroblast populations (Botstein et al., 1982Botstein G.R. Sherer G.K. Leroy E.C. Fibroblast selection in scleroderma. An alternative model of fibrosis.Arthritis Rheum. 1982; 25: 189-195Crossref PubMed Scopus (149) Google Scholar;Goldring et al., 1990Goldring S.R. Stephenson M.L. Downie E. Krane S.M. Korn J.H. Heterogeneity in hormone responses and patterns of collagen synthesis in cloned dermal fibroblasts.J Clin Invest. 1990; 85: 798-803Crossref PubMed Scopus (58) Google Scholar;Jelaska and Korn, 2000Jelaska A. Korn J.H. Role of apoptosis and transforming growth factor beta1 in fibroblast selection and activation in systemic sclerosis.Arthritis Rheum. 2000; 43: 2230-2239Crossref PubMed Scopus (136) Google Scholar). In vitro, SSc skin fibroblasts demonstrate inappropriate growth patterns such as persistent proliferation in serum-free media associated with high expression of the proto-oncogenes, c-myc (Trojanowska et al., 1988Trojanowska M. Wu L.T. LeRoy E.C. Elevated expression of c-myc proto-oncogene in scleroderma fibroblasts.Oncogene. 1988; 3: 477-481PubMed Google Scholar), and c-myb (Piccinini et al., 1996Piccinini G. Luchetti M.M. Caniglia M.L. Carossino A.M. Montroni M. Introna M. Gabrielli A. c-myb proto-oncogene is expressed by quiescent scleroderma fibroblasts and, unlike B-myb gene, does not correlate with proliferation.J Invest Dermatol. 1996; 106: 1281-1286Crossref PubMed Scopus (19) Google Scholar). Furthermore, SSc skin fibroblasts are resistant to Fas-mediated apoptosis that may, in part, be a consequence of exposure to transforming growth factor-β (TGF)-β (Jelaska and Korn, 2000Jelaska A. Korn J.H. Role of apoptosis and transforming growth factor beta1 in fibroblast selection and activation in systemic sclerosis.Arthritis Rheum. 2000; 43: 2230-2239Crossref PubMed Scopus (136) Google Scholar;Santiago et al., 2001Santiago B. Galindo M. Rivero M. Pablos J.L. Decreased susceptibility to Fas-induced apoptosis of systemic sclerosis dermal fibroblasts.Arthritis Rheum. 2001; 44: 1667-1676Crossref PubMed Scopus (69) Google Scholar). One of the most potent pro-fibrotic stimuli to skin fibroblasts in vitro is TGF-β (Denton and Abraham, 2001Denton C.P. Abraham D.J. Transforming growth factor-beta and connective tissue growth factor: Key cytokines in scleroderma pathogenesis.Curr Opin Rheumatol. 2001; 13: 505-511Crossref PubMed Scopus (199) Google Scholar). It has been reported that TGF-β expression in lesional skin of SSc is elevated (Gruschwitz et al., 1990Gruschwitz M. Muller P.U. Sepp N. Hofer E. Fontana A. Wick G. Transcription and expression of transforming growth factor type beta in the skin of progressive systemic sclerosis: A mediator of fibrosis?.J Invest Dermatol. 1990; 94: 197-203Abstract Full Text PDF PubMed Google Scholar;Kulozik et al., 1990Kulozik M. Hogg A. Lankat-Buttgereit B. Krieg T. Co-localization of transforming growth factor beta 2 with alpha 1(I) procollagen mRNA in tissue sections of patients with systemic sclerosis.J Clin Invest. 1990; 86: 917-922Crossref PubMed Scopus (201) Google Scholar;Querfeld et al., 1999Querfeld C. Eckes B. Huerkamp C. Krieg T. Sollberg S. Expression of TGF-beta 1, -beta 2 and -beta 3 in localized and systemic scleroderma.J Dermatol Sci. 1999; 21: 13-22Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), although there are some conflicting findings (Peltonen et al., 1990Peltonen J. Kahari L. Uitto J. Jimenez S.A. Increased expression of type VI collagen genes in systemic sclerosis.Arthritis Rheum. 1990; 33: 1829-1835Crossref PubMed Scopus (94) Google Scholar). Much of the evidence suggesting that TGF-β plays a role in the pathogenesis of SSc is based on in vitro studies of cells in culture (Cotton et al., 1998Cotton S.A. Herrick A.L. Jayson M.I. Freemont A.J. TGF beta—a role in systemic sclerosis?.J Pathol. 1998; 184: 4-6Crossref PubMed Scopus (62) Google Scholar). TGF-β acts as a mitogen for skin fibroblasts, presumably through the induction of PDGF receptors (Ishikawa et al., 1990Ishikawa O. LeRoy E.C. Trojanowska M. Mitogenic effect of transforming growth factor beta 1 on human fibroblasts involves the induction of platelet-derived growth factor alpha receptors.J Cell Physiol. 1990; 145: 181-186Crossref PubMed Scopus (83) Google Scholar). TGF-β induces a marked increase in collagen as well as fibronectin synthesis (Ignotz and Massague, 1986Ignotz R.A. Massague J. Transforming growth factor-beta stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix.J Biol Chem. 1986; 261: 4337-4345Abstract Full Text PDF PubMed Google Scholar;Roberts et al., 1986Roberts A.B. Sporn M.B. Assoian R.K. et al.Transforming growth factor type beta: Rapid induction of fibrosis and angiogenesis invivo and stimulation of collagen formation in vitro.Proc Natl Acad Sci USA. 1986; 83: 4167-4171Crossref PubMed Scopus (2331) Google Scholar). Furthermore, TGF-β induces transdifferentiation of fibroblasts into myofibroblasts (Desmouliere et al., 1993Desmouliere A. Geinoz A. Gabbiani F. Gabbiani G. Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts.J Cell Biol. 1993; 122: 103-111Crossref PubMed Scopus (1748) Google Scholar;Yokozeki et al., 1997Yokozeki M. Moriyama K. Shimokawa H. Kuroda T. Transforming growth factor-beta 1 modulates myofibroblastic phenotype of rat palatal fibroblasts in vitro.Exp Cell Res. 1997; 231: 328-336Crossref PubMed Scopus (59) Google Scholar;Gabbiani, 2003Gabbiani G. The myofibroblast in wound healing and fibrocontractive diseases.J Pathol. 2003; 200: 500-503Crossref PubMed Scopus (1139) Google Scholar), which synthesize elevated levels of collagen and TIMP-1 and show a much stronger effect in response to TGF-β (Kirk and Kahn, 1995Kirk M.D. Kahn A.J. Extracellular matrix synthesized by clonal osteogenic cells is osteoinductive invivo and in vitro: Role of transforming growth factor- beta 1 in osteoblast cell–matrix interaction.J Bone Miner Res. 1995; 10: 1203-1208Crossref PubMed Scopus (26) Google Scholar). TGF-β also stimulates extracellular matrix assembly, including fibrillin assembly (Kissin et al., 2002Kissin E.Y. Lemaire R. Korn J.H. Lafyatis R. Transforming growth factor beta induces fibroblast fibrillin-1 matrix formation.Arthritis Rheum. 2002; 46: 3000-3009Crossref PubMed Scopus (50) Google Scholar), which occurs in associations with myofibroblasts (Kissin and Korn, 2002Kissin E. Korn J.H. Apoptosis and myofibroblasts in the pathogenesis of systemic sclerosis.Curr Rheumatol Rep. 2002; 4: 129-135Crossref PubMed Scopus (36) Google Scholar). We have previously shown that TGF-β exerts its growth and anti-apoptotic effects on fibroblasts, at least in part, by activation of the phosphaditylinositol 3 (PI3) kinase/Akt pathway (Kim et al., 2002Kim G. Jun J.B. Elkon K.B. Necessary role of phosphatidylinositol 3-kinase in transforming growth factor beta-mediated activation of Akt in normal and rheumatoid arthritis synovial fibroblasts.Arthritis Rheum. 2002; 46: 1504-1511Crossref PubMed Scopus (64) Google Scholar). Others have shown similar findings in other cell types (Shin et al., 2001Shin I. Bakin A.V. Rodeck U. Brunet A. Arteaga C.L. Transforming growth factor beta enhances epithelial cell survival via Akt-dependent regulation of FKHRL1.Mol Biol Cell. 2001; 12: 3328-3339Crossref PubMed Scopus (151) Google Scholar;Vinals and Pouyssegur, 2001Vinals F. Pouyssegur J. Transforming growth factor beta1 (TGF-beta1) promotes endothelial cell survival during in vitro angiogenesis via an autocrine mechanism implicating TGF-alpha signaling.Mol Cell Biol. 2001; 21: 7218-7230Crossref PubMed Scopus (172) Google Scholar). Akt is a master regulator of growth control that also influences the function of both the intrinsic (mitochondrial) as well as the extrinsic (death receptor) pathways of apoptosis, through the phosphorylation of its substrates Bad (del Peso et al., 1997del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt.Science. 1997; 278: 687-689Crossref PubMed Scopus (1910) Google Scholar), caspase 9 (Datta et al., 1999Datta S.R. Brunet A. Greenberg M.E. Cellular survival: A play in three Akts.Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3647) Google Scholar), forkhead transcription factors (Brunet et al., 1999Brunet A. Bonni A. Zigmond M.J. et al.Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5151) Google Scholar), and IκB kinase (Ozes et al., 1999Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase.Nature. 1999; 401: 82-85Crossref PubMed Scopus (1834) Google Scholar) resulting in their activation or inhibition. We therefore hypothesized that inappropriate cell growth and survival of skin fibroblasts in SSc were associated with excessive activation of Akt and this phenomenon resulted from stimulation by external factors, such as TGF-β. Here, we report that Akt kinase is activated in SSc skin fibroblasts in vitro and invivo and that exposure of skin fibroblasts to TGF-β-induced Akt activation. Taken together, we propose that TGF-β signaling through Akt contributes to disease pathogenesis. Since TGF-β is associated with resistance of SSc fibroblasts to apoptosis (Jelaska and Korn, 2000Jelaska A. Korn J.H. Role of apoptosis and transforming growth factor beta1 in fibroblast selection and activation in systemic sclerosis.Arthritis Rheum. 2000; 43: 2230-2239Crossref PubMed Scopus (136) Google Scholar;Santiago et al., 2001Santiago B. Galindo M. Rivero M. Pablos J.L. Decreased susceptibility to Fas-induced apoptosis of systemic sclerosis dermal fibroblasts.Arthritis Rheum. 2001; 44: 1667-1676Crossref PubMed Scopus (69) Google Scholar), and we and others have shown that TGF-β can activate the anti-apoptotic kinase, Akt (Bakin et al., 2000Bakin A.V. Tomlinson A.K. Bhowmick N.A. Moses H.L. Arteaga C.L. Phosphatidylinositol 3-kinase function is required for transforming growth factor beta-mediated epithelial to mesenchymal transition and cell migration.J Biol Chem. 2000; 275: 36803-36810Crossref PubMed Scopus (788) Google Scholar;Kim et al., 2002Kim G. Jun J.B. Elkon K.B. Necessary role of phosphatidylinositol 3-kinase in transforming growth factor beta-mediated activation of Akt in normal and rheumatoid arthritis synovial fibroblasts.Arthritis Rheum. 2002; 46: 1504-1511Crossref PubMed Scopus (64) Google Scholar), we performed pilot studies to address whether SSc fibroblasts demonstrated higher Akt activation compared with normal fibroblasts in vitro. Akt activation was determined by western blot analysis using anti-phospho-Akt (p-Akt) antibody and the samples were ranked according to the relative band density ratio of p-Akt to Akt. As shown in Figure 1a, cultured SSc skin fibroblast cell strains showed higher expression of p-Akt compared with normal skin fibroblast cell strains (9.8±0.8 vs 5.6±1.1, p=0.012). As an additional test of Akt activation in cultured SSc fibroblasts in vitro, we used a method that quantifies functional Akt enzymatic activity using glycogen synthase kinase-3 (GSK-3) as the substrate. As shown in Figure 1b, densitometric comparison of Akt activation from three SSc (S4–S6) and four normal (N4–N7) skin fibroblast cell strains showed a borderline significant difference (SSc, 75.2±10.7 vs control, 42.6±7.7, p=0.05, Student's t test). Taken together, these results suggest a higher baseline activation of the Akt kinase in cultured SSc skin fibroblasts compared with normal skin fibroblasts in vitro. Since the in vitro findings were suggestive but not conclusive, we asked whether Akt was activated invivo by examining tissue sections obtained from lesional skin. Control skin sections from inflammatory diseases such as eczema and psoriasis as well as sections from localized scleroderma (morphea) were used as controls. As shown in representative hematoxylin & eosin (H&E)-stained sections in Figure 2a, a much higher proportion of SSc skin fibroblasts stained positive for p-Akt compared with normal control skin sections (compare d and e) and staining was specific as evidenced by lack of staining in SSc fibroblasts with normal rabbit serum (g). As shown under higher magnification (f), many of the p-Akt-positive cells are spindle shaped, indicating their fibroblast origin. A lymphohistocytic inflammatory infiltrate, especially prominent around vessels, observed in the cases of SSc also demonstrated p-Akt-positive staining. For purposes of quantitation, only spindle-shaped nuclei in areas of dermal sclerosis were used for analysis. On average, 210±12 fibroblasts were present and analyzed per sample. A summary of quantitation of p-Akt-positive fibroblasts is shown in Figure 2b. The percentage of p-Akt-positive fibroblasts was significantly higher in SSc patients versus controls (78%±14.0% vs 13%±9%, p<0.001). Since activation of fibroblasts may lead to the production of myofibroblasts in SSc (Kissin and Korn, 2002Kissin E. Korn J.H. Apoptosis and myofibroblasts in the pathogenesis of systemic sclerosis.Curr Rheumatol Rep. 2002; 4: 129-135Crossref PubMed Scopus (36) Google Scholar), we asked whether p-Akt was co-expressed in skin myofibroblasts. In three samples tested, sections were stained for p-Akt and anti-α-smooth muscle actin (SMA). In two samples, p-Akt was positive, but SMA was negative. A third sample stained for both p-Akt and SMA (Figure 3). When the images were merged, there was a clear overlap of the two stains indicating co-expression. TGF-β, IL-4, and CD40 have been implicated in the induction of tissue fibrosis (Kaufman et al., 2001Kaufman J. Graf B.A. Leung E.C. et al.Fibroblasts as sentinel cells: Role of the CDcd40-CDcd40 ligand system in fibroblast activation and lung inflammation and fibrosis.Chest. 2001; 120: 53S-55SCrossref PubMed Scopus (43) Google Scholar;Sime and O'Reilly, 2001Sime P.J. O'Reilly K.M. Fibrosis of the lung and other tissues: New concepts in pathogenesis and treatment.Clin Immunol. 2001; 99: 308-319Crossref PubMed Scopus (281) Google Scholar) and have been shown to induce Akt activation in certain cell types (Andjelic et al., 2000Andjelic S. Hsia C. Suzuki H. Kadowaki T. Koyasu S. Liou H.C. Phosphatidylinositol 3-kinase and NF-kappa B/Rel are at the divergence of CD40-mediated proliferation and survival pathways.J Immunol. 2000; 165: 3860-3867Crossref PubMed Scopus (69) Google Scholar;Bakin et al., 2000Bakin A.V. Tomlinson A.K. Bhowmick N.A. Moses H.L. Arteaga C.L. Phosphatidylinositol 3-kinase function is required for transforming growth factor beta-mediated epithelial to mesenchymal transition and cell migration.J Biol Chem. 2000; 275: 36803-36810Crossref PubMed Scopus (788) Google Scholar;Arron et al., 2001Arron J.R. Vologodskaia M. Wong B.R. Naramura M. Kim N. Gu H. Choi Y. A positive regulatory role for Cbl family proteins in tumor necrosis factor-related activation-induced cytokine (trance) and CD40L-mediated Akt activation.J Biol Chem. 2001; 276: 30011-30017Crossref PubMed Scopus (108) Google Scholar;Kim et al., 2002Kim G. Jun J.B. Elkon K.B. Necessary role of phosphatidylinositol 3-kinase in transforming growth factor beta-mediated activation of Akt in normal and rheumatoid arthritis synovial fibroblasts.Arthritis Rheum. 2002; 46: 1504-1511Crossref PubMed Scopus (64) Google Scholar). To determine whether SSc fibroblasts were hypersensitive to TGF-β signal transduction through the PI3 kinase–Akt pathway, we compared Akt activation between normal (n=3) and SSc (n=3) fibroblasts following incubation with TGF-β in two separate experiments. The ratios for each patient from the two separate experiments were averaged and then compared by Student's t test. The mean±SE for normal (1.7±0.1) and SSc (2.4±0.45) were not significantly different (p=0.19). Although SSc fibroblasts responded to TGF-β by activation of Akt (Figure 4) and were more responsive to TGF-β than normal fibroblasts at 1, 2, 4, and 24 h, the results were not statistically significantly different (not shown). The pathology of SSc is tightly linked to the activation of fibroblast populations (Botstein et al., 1982Botstein G.R. Sherer G.K. Leroy E.C. Fibroblast selection in scleroderma. An alternative model of fibrosis.Arthritis Rheum. 1982; 25: 189-195Crossref PubMed Scopus (149) Google Scholar;Goldring et al., 1990Goldring S.R. Stephenson M.L. Downie E. Krane S.M. Korn J.H. Heterogeneity in hormone responses and patterns of collagen synthesis in cloned dermal fibroblasts.J Clin Invest. 1990; 85: 798-803Crossref PubMed Scopus (58) Google Scholar). Recently, it has been reported that in addition to fibroblast activation, SSc skin fibroblasts are resistant to Fas-mediated apoptosis and this resistance can be enhanced by exposure of the cells to TGF-β (Jelaska and Korn, 2000Jelaska A. Korn J.H. Role of apoptosis and transforming growth factor beta1 in fibroblast selection and activation in systemic sclerosis.Arthritis Rheum. 2000; 43: 2230-2239Crossref PubMed Scopus (136) Google Scholar;Santiago et al., 2001Santiago B. Galindo M. Rivero M. Pablos J.L. Decreased susceptibility to Fas-induced apoptosis of systemic sclerosis dermal fibroblasts.Arthritis Rheum. 2001; 44: 1667-1676Crossref PubMed Scopus (69) Google Scholar). Despite the fact that similar observations have been made in rheumatoid arthritis synovial fibroblasts (Kawakami et al., 1996Kawakami A. Eguchi K. Matsuoka N. Tsuboi M. Kawabe Y. Aoyagi T. Nagataki S. Inhibition of Fas antigen-mediated apoptosis of rheumatoid synovial cells in vitro by transforming growth factor beta 1.Arthritis Rheum. 1996; 39: 1267-1276Crossref PubMed Scopus (75) Google Scholar), the pathway through which TGF-β exerts its protective effect from apoptosis remains unknown. Significantly, the best-characterized pathway of TGF-β signal transduction through Smad 2, 3, and 4 (Roberts and Derynck, 2001Roberts A.B. Derynck R. Meeting report: Signaling schemes for TGF-beta.Sci STKE. 2001; 2001: E43Google Scholar) is not known to directly influence transcription of apoptotic regulators. We recently showed that TGF-β-induced activation of Akt in normal and RA synovial fibroblasts and that TGF-β exerted its anti-apoptotic effect, in part, through the PI3 kinase/Akt pathway (Kim et al., 2002Kim G. Jun J.B. Elkon K.B. Necessary role of phosphatidylinositol 3-kinase in transforming growth factor beta-mediated activation of Akt in normal and rheumatoid arthritis synovial fibroblasts.Arthritis Rheum. 2002; 46: 1504-1511Crossref PubMed Scopus (64) Google Scholar). Since TGF-β plays a prominent role in the pathogenesis of SSc (Simms and Korn, 2002Simms R.W. Korn J.H. Cytokine directed therapy in scleroderma: Rationale, current status, and the future.Curr Opin Rheumatol. 2002; 14: 717-722Crossref PubMed Scopus (43) Google Scholar), we examined whether Akt was activated in skin fibroblasts of SSc patients. Here we show that both by; in vitro and, more significantly, in skin biopsies insitu, Akt is phosphorylated in SSc skin fibroblasts. Although quantitative estimates of expression of phospho-Akt (P-Akt) are not possible by immunohistochemistry, the numbers of fibroblasts expressing p-Akt were strikingly increased in SSc compared with both normal and disease controls. This suggests that Akt phosphorylation is not a non-specific manifestation of any type of skin inflammatory response. The significance of the finding that Akt is activated in SSc skin fibroblasts is severalfold. First, Akt is best known for its anti-apoptotic effect that impacts both the death receptor and mitochondrial pathways (Datta et al., 1999Datta S.R. Brunet A. Greenberg M.E. Cellular survival: A play in three Akts.Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3647) Google Scholar). Resistance to apoptosis, which has been demonstrated experimentally in SSc fibroblasts (Jelaska and Korn, 2000Jelaska A. Korn J.H. Role of apoptosis and transforming growth factor beta1 in fibroblast selection and activation in systemic sclerosis.Arthritis Rheum. 2000; 43: 2230-2239Crossref PubMed Scopus (136) Google Scholar;Santiago et al., 2001Santiago B. Galindo M. Rivero M. Pablos J.L. Decreased susceptibility to Fas-induced apoptosis of systemic sclerosis dermal fibroblasts.Arthritis Rheum. 2001; 44: 1667-1676Crossref PubMed Scopus (69) Google Scholar), presumably contributes to accumulation of activated fibroblasts in the skin promoting the accumulation of extracellular matrix. Second, Akt has been implicated in cell transdifferentiation such as epithelial to mesenchymal transition (Bakin et al., 2000Bakin A.V. Tomlinson A.K. Bhowmick N.A. Moses H.L. Arteaga C.L. Phosphatidylinositol 3-kinase function is required for transforming growth factor beta-mediated epithelial to mesenchymal transition and cell migration.J Biol Chem. 2000; 275: 36803-36810Crossref PubMed Scopus (788) Google Scholar). Increased myofibroblasts are a key finding in SSc (Jelaska and Korn, 2000Jelaska A. Korn J.H. Role of apoptosis and transforming growth factor beta1 in fibroblast selection and activation in systemic sclerosis.Arthritis Rheum. 2000; 43: 2230-2239Crossref PubMed Scopus (136) Google Scholar) and they are associated with potent fibrogenic activity (Gabbiani, 1981Gabbiani G. The myofibroblast: A key cell for wound healing and fibrocontractive diseases.Prog Clin Biol Res. 1981; 54: 183-194PubMed Google Scholar;Kirk et al., 1995Kirk T.Z. Mark M.E. Chua C.C. Chua B.H. Mayes M.D. Myofibroblasts from scleroderma skin synthesize elevated levels of collagen and tissue inhibitor of metalloproteinase (TIMP-1) with two forms of TIMP-1.J Biol Chem. 1995; 270: 3423-3428Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In a small sampling of skin biopsies from SSc patients, we were able to demonstrate co-expression of activated Akt and α-SMA, consistent with the notion that Akt activation may play a role in the generation of these myofibroblasts in disease states. TGF-β has been implicated in the conversion of fibroblasts to myofibroblasts (Desmouliere et al., 1993Desmouliere A. Geinoz A. Gabbiani F. Gabbiani G. Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts.J Cell Biol. 1993; 122: 103-111Crossref PubMed Scopus (1748) Google Scholar;Yokozeki et al., 1997Yokozeki M. Moriyama K. Shimokawa H. Kuroda T. Transforming growth factor-beta 1 modulates myofibroblastic phenotype of rat palatal fibroblasts in vitro.Exp Cell Res. 1997; 231: 328-336Crossref PubMed Scopus (59) Google Scholar), a transformation that occurs in concert with induction of apoptosis resistance (Jelaska and Korn, 2000Jelaska A. Korn J.H. Role of apoptosis and transforming growth factor beta1 in fibroblast selection and activation in systemic sclerosis.Arthritis Rheum. 2000; 43: 2230-2239Crossref PubMed Scopus (136) Google Scholar). Although the pathway whereby this occurs is not known, several observations lead us to speculate that this may occur through the PI3 kinase/Akt pathway. PI3 kinase plays an important role in smooth muscle differentiation (Hayashi et al., 1998Hayashi K. Saga H. Chimori Y. Kimura K. Yamanaka Y. Sobue K. Differentiated phenotype of smooth muscle cells depends on signaling pathways through insulin-like growth factors and phosphatidylinositol 3-kinase.J Biol Chem. 1998; 273: 28860-28867Crossref PubMed Scopus (150) Google Scholar) and myogenic signaling of PI3 kinase requires Akt kinase (Jiang et al., 1999Jiang B.H. Aoki M. Zheng J.Z. Li J. Vogt P.K. Myogenic signaling of phosphatidylinositol 3-kinase requires the serine-threonine kinase Akt/protein kinase B.Proc Natl Acad Sci USA. 1999; 96: 2077-2081Crossref PubMed Scopus (215) Google Scholar). Furthermore, apigenin, a common dietary flavonoid, which inhibited Akt activation, decreased expression of the myofibroblast phenotype (Ricupero et al., 2001Ricupero D.A. Poliks C.F. Rishikof D.C. Cuttle K.A. Kuang P.P. Goldstein R.H. Phosphatidylinositol 3-kinase-dependent stabilization of alpha1(I) collagen mRNA in human lung fibroblasts.Am J Physiol Cell Physiol. 2001; 281: C99-C105PubMed Google Scholar). There are several potential explanations for activation of Akt in SSc fibroblasts insitu. The most straightforward explanation is that dermal fibroblasts are exposed to increased amounts of bioactive TGF-β in lesional tissue. As discussed above, however, there remains controversy regarding the levels of active TGF-β at sites of fibrosis in patients with this disease. An alternative explanation is that patients are hypersensitive to the effects of TGF-β. At a biochemical level, this could be explained by failure to downmodulate TGF-β signal transduction, as has recently been reported for TGF-β signaling through the Smad pathway (Dong et al., 2002Dong C. Zhu S. Wang T. et al.Deficient Smad7 expression: A putative molecular defect in scleroderma.Proc Natl Acad Sci USA. 2002; 99: 3908-3913Crossref PubMed Scopus (226) Google Scholar). The TGF-β-associated antagonist LAP (latency-associated peptide) has been shown to decrease skin fibrosis in a mouse model of scleroderma (Zhang et al., 2003Zhang Y. McCormick L.L. Gilliam A.C. Latency-associated peptide prevents skin fibrosis in murine sclerodermatous graft-versus-host disease, a model for human scleroderma.J Invest Dermatol. 2003; 121: 713-719Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), suggesting that regulation of TGF-β activity rather than absolute levels of TGF-β contribute to the fibrosis in this disease. Many of the genes reported as overexpressed in SSc fibroblasts are known to be TGF-β-inducible genes including CTGF, collagens, and fibronectin. Gene array studies support the concept that there is overlap between TGF-β-induced genes and those that characterize scleroderma fibroblasts (Verrecchia et al., 2001Verrecchia F. Chu M.L. Mauviel A. Identification of novel TGF-beta/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach.J Biol Chem. 2001; 276: 17058-17062Crossref PubMed Scopus (508) Google Scholar; Joseph H. Korn, unpublished observations). The increased proportion of myofibroblasts in SSc may be another manifestation of this property. Although we observed TGF-β to be a potent activator of Akt in SSc-cultured fibroblasts, Akt activation in response to TGF-β was not statistically significantly different between SSc and normal controls. Although this result suggests that there is no intrinsic abnormality of the Akt pathway in SSc fibroblasts, cultured SSc skin fibroblasts are reported to be less responsive to TGF-βin vitro (Kikuchi et al., 1992Kikuchi K. Hartl C.W. Smith E.A. LeRoy E.C. Trojanowska M. Direct demonstration of transcriptional activation of collagen gene expression in systemic sclerosis fibroblasts: Insensitivity to TGF beta 1 stimulation.Biochem Biophys Res Commun. 1992; 187: 45-50Crossref PubMed Scopus (85) Google Scholar) resulting from chronic exposure to TGF-βinvivo. More detailed studies, possibly with fibroblasts from uninvolved skin, will be needed to determine whether the PI3 kinase/Akt pathway, including the key phosphatases that downmodulate p-Akt, PTEN, and SHIP (Datta et al., 1999Datta S.R. Brunet A. Greenberg M.E. Cellular survival: A play in three Akts.Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3647) Google Scholar), function normally in this disease. In addition to TGF-β, other cytokines, growth factors, or ligands such as CD40L could potentially activate Akt and deserve further study. A final point to note is that attenuation of PI3 kinase activity by specific inhibitors decreased stabilization of α-1 (I) collagen mRNA in human lung fibroblasts and that Akt activation correlated with the change in α-1 (I) collagen mRNA levels (Ricupero et al., 2001Ricupero D.A. Poliks C.F. Rishikof D.C. Kuang P.P. Goldstein R.H. Apigenin decreases expression of the myofibroblast phenotype.FEBS Lett. 2001; 506: 15-21Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Since SSc skin fibroblasts are characterized by increased collagen production due, in large part, to increased mRNA stability (Eckes et al., 1996Eckes B. Mauch C. Huppe G. Krieg T. Differential regulation of transcription and transcript stability of pro-alpha 1(I) collagen and fibronectin in activated fibroblasts derived from patients with systemic scleroderma.Biochem J. 1996; 315: 549-554Crossref PubMed Scopus (51) Google Scholar), the PI3 kinase/Akt pathway may be more directly involved in SSc pathogenesis than hitherto thought. Whether selective blockade of this pathway can be achieved without disruption of normal skin function remains a relevant avenue for future research. Recombinant human TGF-β1 was purchased from R&D System (Minneapolis, Minnesota). Antibodies to Akt, phospho-specific Akt (Ser473), and phospho-Akt (Ser473) immunohistochemistry-specific antibodies were purchased from Cell Signaling Technology (Beverly, Massachusetts) and to anti-α-SMA from Beckman Coulter (Fullerton, California). Horse anti-rabbit IgG antibody was from Jackson ImmunoResearch Laboratories (West Grove, Pennsylvania). A total of 15 SSc patients (average age 50 y, 80% Caucasian and 80% female) were included in this study. All SSc patients had diffuse disease and fulfilled the American College of Rheumatology criteria for SSc (Masi et al., 1980Masi A.J. Rodnan G.P. Medsger Jr., J.A. et al.Preliminary criteria for the classification of systemic sclerosis (scleroderma).Arthritis Rheum. 1980; 23: 581-590Crossref PubMed Scopus (4431) Google Scholar). Skin biopsies were obtained from patients and control subjects after informed consent in a protocol approved by the Institutional Review Board for Human Studies. Control biopsies of lesional skin were obtained from patients with psoriasis, eczema, and morphea. The tissue was fixed in formalin, embedded in paraffin, and sections were stained with H&E. Fibroblasts were isolated and cell strains cultured as described previously (Jelaska and Korn, 2000Jelaska A. Korn J.H. Role of apoptosis and transforming growth factor beta1 in fibroblast selection and activation in systemic sclerosis.Arthritis Rheum. 2000; 43: 2230-2239Crossref PubMed Scopus (136) Google Scholar). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco Invitrogen, Grand Island, New York) supplemented with 10% fetal calf serum (FCS, Gibco BRL, Rockville, Maryland), 1% penicillin and streptomycin and L-glutamine (Gibco BRL) in a humidified 5% CO2, 95% air incubator at 37°C. Skin fibroblasts at third to seventh passages were used for the experiments. Cells were seeded on six-well plates in DMEM with 10% FCS overnight. The cultures were then starved overnight in serum-free medium (DMEM with penicillin/streptomycin and L-glutamine) to determine baseline Akt activation (Kim et al., 2002Kim G. Jun J.B. Elkon K.B. Necessary role of phosphatidylinositol 3-kinase in transforming growth factor beta-mediated activation of Akt in normal and rheumatoid arthritis synovial fibroblasts.Arthritis Rheum. 2002; 46: 1504-1511Crossref PubMed Scopus (64) Google Scholar). In some experiments, cells were cultured in the presence of TGF-β1 (5 ng per mL) for 1 h. Cells were washed twice in ice-cold PBS and lysed with lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM Na3VO4, 0.1% 2-mercaptoethanol, 1% Triton X-100, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM sodium β-glycerophosphate, 0.1 mM PMSF, 1 mg per mL of aprotinin, leupeptin, and pepstatin, 137 mM NaCl, and 10% glycerol). Protein concentration of cell lysates was determined by the Bradford assay kit (Bio-Rad Laboratories, Hercules, California). Proteins (50 μg) were resolved by SDS-PAGE (10%) and blotted to nitrocellulose membranes as described (Kim et al., 2002Kim G. Jun J.B. Elkon K.B. Necessary role of phosphatidylinositol 3-kinase in transforming growth factor beta-mediated activation of Akt in normal and rheumatoid arthritis synovial fibroblasts.Arthritis Rheum. 2002; 46: 1504-1511Crossref PubMed Scopus (64) Google Scholar). Rabbit anti-Akt (1:1000) or anti-phospho-specific (Ser 473) Akt (1:1000) primary antibodies and horse anti-rabbit IgG (1:1000) secondary antibody were used as probes and antibody binding was detected by the enhanced chemiluminescent kit (Amersham Pharmacia Biotech, Piscataway, New Jersey) as recommended by the manufacturer. Band density was quantified by densitometry using the ImageQuant program (Molecular Dynamics, Sunnyvale, California). A functional assay for Akt phosphorylation of the substrate GSK-3 was performed using a kit from New England Biolabs (Beverly, Massachusetts) according to the manufacturer's recommendations. Briefly, 1 × 106 cells were starved overnight in serum-free medium. Cells were lysed and phospho-Akt immunoprecipitated from cell extracts (500 μg) with immobilized phospho-specific Akt (Ser473) antibody. The bound enzyme was incubated with the substrate, GSK-3 fusion protein, in the presence of 200 mM ATP and kinase buffer (25 mM Tris-HCl, pH 7.5, 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2). The extent of GSK-3 phosphorylation was determined by immunoblotting with anti-phospho-specific GSK-3 antibody and quantified by densitometry. The band density obtained with the test samples were compared with the positive control (normal skin fibroblasts stimulated with 10% fetal bovine serum overnight) and expressed as a percentage (test sample/positive control × 100). Formalin-fixed, paraffin-embedded skin samples were sectioned for immunohistochemistry. Six-micron sections were treated with 10 mM sodium citrate, 96°C × 15 min for antigen retrieval, stained with primary antibody (phospho-Akt 1:25 for 2 h), followed by the appropriate secondary biotinylated IgG, amplified with immunoperoxidase (Vectastain ABC kit, Vector Laboratories, Burlingame, California), and visualized with diaminobenzidine as chromagen. Sections were counterstained with hematoxylin to visualize tissue morphology. For immunofluorescence, 6 μm sections were treated with 10 mM sodium citrate, 96°C × 15 min, stained with anti-p-Akt (1:25 for 2 h), followed by FITC-conjugated anti-rabbit antibody (1:400, Vector Laboratories). Sections were then stained with anti-α-SMA antibody (1:2000 for 2 h), followed by Texas Red-conjugated anti-mouse antibody (1:800, Vector Laboratories). DAPI, 50 μg per mL (Sigma), was used to visualize cell nuclei. All antibody incubations were performed at room temperature. Image acquisition was performed using a Nikon Microphot-SA microscope equipped with a CCD camera (Photometrics, Tuscon, Arizona). Data were collected as single 1024 bit images, and RGB color merged using the software program IPLab (Signal Analytics, Vienna, Virginia). Five fields were arbitrarily chosen and counted per specimen at 20′ (1.070 mm2). The total fibroblast numbers were counted on H&E-stained sections and fibroblasts positive for peroxidase staining were counted in the same fields. Results are expressed as a percentage (phospho-Akt-positive fibroblasts/total fibroblasts in the field × 100). Values presented are mean±standard error of mean (SEM). The statistical analyses were performed using Student's t test. Differences with p-values <0.05 were considered statistically significant. This work was supported by the Scleroderma Foundation (K. B. E.), Dermatology Foundation (M. K.), the National Institutes of Health NIAMS RO1 AR32343 (J. H. K.), and Hanyang University (J.B.J.).
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