- Email: [email protected]
Wound healing and TGF_β expression in TIEG knockout mice
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
the progression of renal fibrosis. When taken together with our results, this suggests a novel function for SFRP4, as a putative regulator of the cytokine cascade that leads to skin fibrosis in Tsk mice and SSc, by controlling the extent of the Wnt activation. doi:10.1016/j.matbio.2006.08.096
74 Suppression of Fli1 activates profibrotic gene program in vivo M. Markiewicz, Y. Asano, W.K. Dennis, T. Maria Medical University of SC, Charleston SC 29425, United States The excessive deposition of collagen is the key feature of fibrosis, including scleroderma, however the molecular mechanism of fibrosis remains poorly understood. Previously published studies have implicated Ets family member, Fli1, in collagen and CCN2 gene repression. Furthermore, Fli1 is consistently reduced in the skin of patients with scleroderma possibly via epigenetic mechanisms. The goal of this study was to determine the role of Fli1 in collagen biosynthesis and fibrillogenesis in vivo using two mouse models, heterozygous Fli1 mice (Fli1+/−) and mice with the homozygous deletion of C-terminal domain of Fli1 gene (Fli1 rec/rec). Mice with reduced levels of Fli1 gene showed abnormal collagen biosynthesis and ultrastructure. The changes were more severe in Fli1 rec/rec mice, suggesting an important role for the Cterminal domain of Fli1 in collagen regulation. The mRNA levels of interstitial collagens, including type I, II, III and V were dramatically increased in the skin tissues of Fli1 mutant mice. Similarly, collagen protein levels were increased as demonstrated by acetic acid extraction of newly synthesized collagen and by western blot of skin extracts. Ultrastructural abnormalities included the presence of immature thin collagen fibrils as well as the very thick irregularly shaped fibrils. Abnormal fibrillogenesis correlated with the increased expression of PLOD2, and reduced levels of decorin, fibromodulin and lumican mRNAs. Together, our data show that Fli1 is a key regulator of the collagen homeostasis in the skin. Reduced levels of Fli1 mimic the main features of scleroderma, thus, supporting the view that the absence of Fli1 plays a pathological role in scleroderma skin fibrosis. doi:10.1016/j.matbio.2006.08.097
75 Shed E-cadherin controls influx of CD103+dendritic cells J.K. McGuire, A.M. Manicone
S35
University of Washington, Seattle, WA 98109, United States Chronic inflammation is an important contributor to fibrosis, and T-lymphocytes and dendritic cells (DC) that express the E-cadherin-binding αEβ7-integrin (CD103) are increased in human pulmonary fibrosis. Matrilysin-mediated shedding of E-cadherin (E-cad) is essential for epithelial repair, and a lack of E-cad shedding in matrilysin-null (mmp7−/−) mice correlates with diminished pulmonary fibrosis after bleomycin-induced injury. To assess if matrilysin-mediated E-cad shedding regulates CD103 + leukocyte influx, we analyzed subpopulations of CD103+-cells in lung tissue and bronchoalveolar lavage (BAL) of wildtype (WT) and mmp7−/− mice after bleomycin. During the initiation of fibrosis (day 7–10), we observed a marked increase in the numbers of CD103+ DC (CD11c+, F4/80−) and T-cells (CD3+) in the sub-epithelial compartment around injured airways and alveoli in WT mice compared to mmp7−/− mice, in which CD103+ cells were fewer in number and largely constrained to the perivascular space. At 14–21 days, CD103+ cells were found in and around areas of organized fibrosis, and inflammatory cells in BAL were primarily lymphocytes (55–70% of total WBC). Whereas the percent of CD103+ T-cells did not differ between genotypes, the percent of CD103+ DC was significantly higher in WT compared to mmp7−/− BAL. Our data suggest that matrilysindependent E-cad shedding is not only needed for normal repair but also contributes to the pathogenesis of pulmonary fibrosis by specifically regulating the recruitment of CD103+ DC into the lung and airspaces. We are now assessing how matrilysingenerated soluble E-cad directly regulates CD103+ DC recruitment and activation in chronic lung injury. doi:10.1016/j.matbio.2006.08.098
76 Wound healing and TGF-β expression in TIEG knockout mice S.L. Moran, M. Taguchi, P.C. Amadio, K. An Mayo Clinic School of Medicine, Rochester, MN, 55905, United States Background: Although TGF-β has a broad effect on wound healing, the exact role for TGF-β during wound healing remains unclear. TGF-β Inducible Early Gene (TIEG), a primary response gene for TGF-β, controls the activity of the TGF-β/ Smad pathway, a main primary TGF-β signaling pathway. The purpose of this study was to investigate the role of TIEG in wound healing using TIEG knockout mice. Methods: Thirty C57Black/129 mice, consisting of 15 TIEG knockout mice (TIEG−/−) and 15 wild type control mice (TIEG +/+) were used. A 2-cm transverse, linear, full-thickness incision symmetrically on the mouse back was made. Five
S36
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
mice of each group were sacrificed on the designated day (postoperative day 3, 7 and 14) and wound breaking strengths were measured. The specimens were also analyzed by histology and Western blot. Results: Although there was no significant difference in wound breaking strength between the TIEG−/− mice and controls at postoperative days 3 and 7, and both groups showed similar re-epithelialization at day 7, the TIEG−/− mice had a significantly lower wound breaking strength than the controls at day 14 (p<0.01). In Western blot analysis, increased expression of TGF-β2 and TGF-β3 was observed in the TIEG−/− mice at days 3 and 7, and day 7, respectively. Conclusion: In this study, we demonstrated that an absence of TIEG had significant effects on the healing of full-thickness incisional wounds, with decreased wound breaking strength at post operative day 14 as well in alterations in TGF-β production when compared to control mice. The results suggest that TIEG expression is an important factor needed to initiate and sustain normal cutaneous wound healing. doi:10.1016/j.matbio.2006.08.099
77 FRNK modulates lung fibroblast migration and lung fibrosis Q. Ding, K.E. White, T. Jin, C.L. Gladson, M.A. Olman Univ. of Alabama at Birmingham, AL 35294, United States RATIONALE: Focal adhesion kinase (FAK) plays a critical role in cell migration. FAK related non-kinase (FRNK) is a dominant inhibitor of FAK-dependent cell migration. We therefore hypothesized that FRNK modulates lung fibrosis in vivo through inhibition of lung fibroblast migration. METHODS: The effect of gain of function of FRNK (adenovirus-mediated overexpression) on fibroblast migration was examined by a monolayer wound healing assay. The fibrotic response to Bleomycin was compared in heterozygous FRNK-knockout mice with that in congenic wild type mice. RESULTS: IPF-patient derived lung fibroblasts show increased migration rates, and concordant increased FAK activation and decreased FRNK protein levels, compared to that in normal human lung fibroblasts. FRNK overexpression inhibits both basal and pro-fibrotic factor (PDGF-BB and TGF-β1)-stimulated fibroblast migration, and FAK activation, in primary human lung fibroblasts derived from both IPF patients and non-fibrotic controls. FRNK-knockout mice (heterozygotes) demonstrate no difference in proliferation, angiogenesis, or inflammation after intratracheal Bleomycin, when compared with congenic wild type mice. However, FRNK knockout mice show greatly increased sensitivity to the
pro-fibrotic effects (e.g., 2-fold greater lung hydroxyproline, 5-fold greater lesional volume) of Bleomycin. CONCLUSIONS: The data demonstrate that FRNK is a key inhibitor of lung fibroblast migration in vitro, and a modulator of lung fibrosis in vivo. (Supported by UAB grant and CAMRC, and AHA to Q Ding, and by NHLBI and VA Merit Award to MA Olman). doi:10.1016/j.matbio.2006.08.100
78 IL-1β alters fibroblast proliferation through PGE2 via EP3 K. White a, Q. Ding a, M. Peters-Golden b, B. Moore b, L. Ware c , M.A. Matthay d, M.A. Olman a a
University of Alabama at Birmingham, United States University of Michigan, United States c Vanderbilt University, United States d University of California at San Francisco, United States b
We recently published that interleukin-1β (IL-1β) is a key mediator of ALI edema fluid-induced fibroblast proliferation. A study of IL-1β physiological responses in the PGE2 pathway was undertaken.Human lung fibroblasts were exposed to ALI and hydrostatic edema patient-derived pulmonary edema fluid ± agonists/antagonists of IL1-B, or the EP2- or EP3-PGE2 receptors. The expression of COX products and proliferation rates were determined. Exogenous IL-1β induced COX-2 mRNA (but not COX-1), and PGE2 production in a dose and serum-dependent manner. Blocking studies of edema fluid with IL-1 receptor antagonist indicate that IL-1β is the major COX-2 mRNA/PGE2-inducing factor in ALI edema fluid, and that IL-1β accounts for the differential PGE2 response to edema fluid from patients with hydrostatic edema. Exogenously added, purified PGE2 was surprisingly both pro-mitogenic (@10-8M) and antiproliferative (@10-6M) over the physiologic range, and the PGE2 concentrations in ALI edema fluid were 5-fold greater than that in hydrostatic edema fluid. Whereas the EP3 selective agonist, sulprostone, had pro-mitogenic and cAMPdecreasing effecs, the EP2 selective receptor agonist, butaprost, was anti-proliferative and cAMP-inducing, as was forskolin. The relative levels of PGE2 receptors in fibroblasts were EP3>EP2>>EP4>EP1 by Western blotting. These data suggest that the IL-1β in ALI edema fluid induces fibroblast PGE2 production, which alters fibroblast proliferation in an autocrine manner through the predominant PGE2 receptor, EP3. This represents a novel fibroproliferative pathway in acute lung injury. doi:10.1016/j.matbio.2006.08.101
the progression of renal fibrosis. When taken together with our results, this suggests a novel function for SFRP4, as a putative regulator of the cytokine cascade that leads to skin fibrosis in Tsk mice and SSc, by controlling the extent of the Wnt activation. doi:10.1016/j.matbio.2006.08.096
74 Suppression of Fli1 activates profibrotic gene program in vivo M. Markiewicz, Y. Asano, W.K. Dennis, T. Maria Medical University of SC, Charleston SC 29425, United States The excessive deposition of collagen is the key feature of fibrosis, including scleroderma, however the molecular mechanism of fibrosis remains poorly understood. Previously published studies have implicated Ets family member, Fli1, in collagen and CCN2 gene repression. Furthermore, Fli1 is consistently reduced in the skin of patients with scleroderma possibly via epigenetic mechanisms. The goal of this study was to determine the role of Fli1 in collagen biosynthesis and fibrillogenesis in vivo using two mouse models, heterozygous Fli1 mice (Fli1+/−) and mice with the homozygous deletion of C-terminal domain of Fli1 gene (Fli1 rec/rec). Mice with reduced levels of Fli1 gene showed abnormal collagen biosynthesis and ultrastructure. The changes were more severe in Fli1 rec/rec mice, suggesting an important role for the Cterminal domain of Fli1 in collagen regulation. The mRNA levels of interstitial collagens, including type I, II, III and V were dramatically increased in the skin tissues of Fli1 mutant mice. Similarly, collagen protein levels were increased as demonstrated by acetic acid extraction of newly synthesized collagen and by western blot of skin extracts. Ultrastructural abnormalities included the presence of immature thin collagen fibrils as well as the very thick irregularly shaped fibrils. Abnormal fibrillogenesis correlated with the increased expression of PLOD2, and reduced levels of decorin, fibromodulin and lumican mRNAs. Together, our data show that Fli1 is a key regulator of the collagen homeostasis in the skin. Reduced levels of Fli1 mimic the main features of scleroderma, thus, supporting the view that the absence of Fli1 plays a pathological role in scleroderma skin fibrosis. doi:10.1016/j.matbio.2006.08.097
75 Shed E-cadherin controls influx of CD103+dendritic cells J.K. McGuire, A.M. Manicone
S35
University of Washington, Seattle, WA 98109, United States Chronic inflammation is an important contributor to fibrosis, and T-lymphocytes and dendritic cells (DC) that express the E-cadherin-binding αEβ7-integrin (CD103) are increased in human pulmonary fibrosis. Matrilysin-mediated shedding of E-cadherin (E-cad) is essential for epithelial repair, and a lack of E-cad shedding in matrilysin-null (mmp7−/−) mice correlates with diminished pulmonary fibrosis after bleomycin-induced injury. To assess if matrilysin-mediated E-cad shedding regulates CD103 + leukocyte influx, we analyzed subpopulations of CD103+-cells in lung tissue and bronchoalveolar lavage (BAL) of wildtype (WT) and mmp7−/− mice after bleomycin. During the initiation of fibrosis (day 7–10), we observed a marked increase in the numbers of CD103+ DC (CD11c+, F4/80−) and T-cells (CD3+) in the sub-epithelial compartment around injured airways and alveoli in WT mice compared to mmp7−/− mice, in which CD103+ cells were fewer in number and largely constrained to the perivascular space. At 14–21 days, CD103+ cells were found in and around areas of organized fibrosis, and inflammatory cells in BAL were primarily lymphocytes (55–70% of total WBC). Whereas the percent of CD103+ T-cells did not differ between genotypes, the percent of CD103+ DC was significantly higher in WT compared to mmp7−/− BAL. Our data suggest that matrilysindependent E-cad shedding is not only needed for normal repair but also contributes to the pathogenesis of pulmonary fibrosis by specifically regulating the recruitment of CD103+ DC into the lung and airspaces. We are now assessing how matrilysingenerated soluble E-cad directly regulates CD103+ DC recruitment and activation in chronic lung injury. doi:10.1016/j.matbio.2006.08.098
76 Wound healing and TGF-β expression in TIEG knockout mice S.L. Moran, M. Taguchi, P.C. Amadio, K. An Mayo Clinic School of Medicine, Rochester, MN, 55905, United States Background: Although TGF-β has a broad effect on wound healing, the exact role for TGF-β during wound healing remains unclear. TGF-β Inducible Early Gene (TIEG), a primary response gene for TGF-β, controls the activity of the TGF-β/ Smad pathway, a main primary TGF-β signaling pathway. The purpose of this study was to investigate the role of TIEG in wound healing using TIEG knockout mice. Methods: Thirty C57Black/129 mice, consisting of 15 TIEG knockout mice (TIEG−/−) and 15 wild type control mice (TIEG +/+) were used. A 2-cm transverse, linear, full-thickness incision symmetrically on the mouse back was made. Five
S36
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
mice of each group were sacrificed on the designated day (postoperative day 3, 7 and 14) and wound breaking strengths were measured. The specimens were also analyzed by histology and Western blot. Results: Although there was no significant difference in wound breaking strength between the TIEG−/− mice and controls at postoperative days 3 and 7, and both groups showed similar re-epithelialization at day 7, the TIEG−/− mice had a significantly lower wound breaking strength than the controls at day 14 (p<0.01). In Western blot analysis, increased expression of TGF-β2 and TGF-β3 was observed in the TIEG−/− mice at days 3 and 7, and day 7, respectively. Conclusion: In this study, we demonstrated that an absence of TIEG had significant effects on the healing of full-thickness incisional wounds, with decreased wound breaking strength at post operative day 14 as well in alterations in TGF-β production when compared to control mice. The results suggest that TIEG expression is an important factor needed to initiate and sustain normal cutaneous wound healing. doi:10.1016/j.matbio.2006.08.099
77 FRNK modulates lung fibroblast migration and lung fibrosis Q. Ding, K.E. White, T. Jin, C.L. Gladson, M.A. Olman Univ. of Alabama at Birmingham, AL 35294, United States RATIONALE: Focal adhesion kinase (FAK) plays a critical role in cell migration. FAK related non-kinase (FRNK) is a dominant inhibitor of FAK-dependent cell migration. We therefore hypothesized that FRNK modulates lung fibrosis in vivo through inhibition of lung fibroblast migration. METHODS: The effect of gain of function of FRNK (adenovirus-mediated overexpression) on fibroblast migration was examined by a monolayer wound healing assay. The fibrotic response to Bleomycin was compared in heterozygous FRNK-knockout mice with that in congenic wild type mice. RESULTS: IPF-patient derived lung fibroblasts show increased migration rates, and concordant increased FAK activation and decreased FRNK protein levels, compared to that in normal human lung fibroblasts. FRNK overexpression inhibits both basal and pro-fibrotic factor (PDGF-BB and TGF-β1)-stimulated fibroblast migration, and FAK activation, in primary human lung fibroblasts derived from both IPF patients and non-fibrotic controls. FRNK-knockout mice (heterozygotes) demonstrate no difference in proliferation, angiogenesis, or inflammation after intratracheal Bleomycin, when compared with congenic wild type mice. However, FRNK knockout mice show greatly increased sensitivity to the
pro-fibrotic effects (e.g., 2-fold greater lung hydroxyproline, 5-fold greater lesional volume) of Bleomycin. CONCLUSIONS: The data demonstrate that FRNK is a key inhibitor of lung fibroblast migration in vitro, and a modulator of lung fibrosis in vivo. (Supported by UAB grant and CAMRC, and AHA to Q Ding, and by NHLBI and VA Merit Award to MA Olman). doi:10.1016/j.matbio.2006.08.100
78 IL-1β alters fibroblast proliferation through PGE2 via EP3 K. White a, Q. Ding a, M. Peters-Golden b, B. Moore b, L. Ware c , M.A. Matthay d, M.A. Olman a a
University of Alabama at Birmingham, United States University of Michigan, United States c Vanderbilt University, United States d University of California at San Francisco, United States b
We recently published that interleukin-1β (IL-1β) is a key mediator of ALI edema fluid-induced fibroblast proliferation. A study of IL-1β physiological responses in the PGE2 pathway was undertaken.Human lung fibroblasts were exposed to ALI and hydrostatic edema patient-derived pulmonary edema fluid ± agonists/antagonists of IL1-B, or the EP2- or EP3-PGE2 receptors. The expression of COX products and proliferation rates were determined. Exogenous IL-1β induced COX-2 mRNA (but not COX-1), and PGE2 production in a dose and serum-dependent manner. Blocking studies of edema fluid with IL-1 receptor antagonist indicate that IL-1β is the major COX-2 mRNA/PGE2-inducing factor in ALI edema fluid, and that IL-1β accounts for the differential PGE2 response to edema fluid from patients with hydrostatic edema. Exogenously added, purified PGE2 was surprisingly both pro-mitogenic (@10-8M) and antiproliferative (@10-6M) over the physiologic range, and the PGE2 concentrations in ALI edema fluid were 5-fold greater than that in hydrostatic edema fluid. Whereas the EP3 selective agonist, sulprostone, had pro-mitogenic and cAMPdecreasing effecs, the EP2 selective receptor agonist, butaprost, was anti-proliferative and cAMP-inducing, as was forskolin. The relative levels of PGE2 receptors in fibroblasts were EP3>EP2>>EP4>EP1 by Western blotting. These data suggest that the IL-1β in ALI edema fluid induces fibroblast PGE2 production, which alters fibroblast proliferation in an autocrine manner through the predominant PGE2 receptor, EP3. This represents a novel fibroproliferative pathway in acute lung injury. doi:10.1016/j.matbio.2006.08.101