Role of B7H3/IL-33 Signaling in Pulmonary Fibrosis-induced Profibrogenic Alterations in Bone Marrow.

Rationale: The impact of lung insult on the bone marrow (BM) and subsequent disease is unknown.Objectives: To study alterations in the BM in response to lung injury/fibrosis and examine their impact on subsequent lung insult.Methods: BM cells from control or bleomycin-treated donor mice were transplanted into naive mice, which were subsequently evaluated for bleomycin-induced pulmonary fibrosis. In addition, the effect of prior bleomycin treatment on subsequent fibrosis was examined in wild-type and B7H3-knockout mice. Samples from patients with idiopathic pulmonary fibrosis were analyzed for potential clinical relevance of the findings.Measurements and Main Results: Recipient mice transplanted with BM from bleomycin-pretreated donors showed significant exacerbation of subsequent fibrosis with increased B7H3+ cell numbers and a T-helper cell type 2-skewed phenotype. Pretreatment with a minimally fibrogenic/nonfibrogenic dose of bleomycin also caused exacerbation, but not in B7H3-deficient mice. Exacerbation was not observed if the mice received naive BM cell transplant after the initial bleomycin pretreatment. Soluble B7H3 stimulated BM Ly6Chi monocytic cell expansion in vitro and caused similar expansion in the lung in vivo. Notably, soluble B7H3 was elevated in plasma of patients with idiopathic pulmonary fibrosis and in BAL fluid in those with acute exacerbation. Finally, ST2 deficiency diminished the bleomycin-induced B7H3 and IL-13 upregulation, suggesting a role for type 2 innate lymphoid cells.Conclusions: Pulmonary fibrosis caused significant alterations in BM with expansion and activation of monocytic cells, which enhanced fibrosis when transplanted to naive recipients with potential mediation by a novel role for B7H3 in the pathophysiology of pulmonary fibrosis in both mice and humans.

Bone Marrow CD11c+ Cell-Derived Amphiregulin Promotes Pulmonary Fibrosis.

Amphiregulin (AREG), an epidermal growth factor receptor ligand, is implicated in tissue repair and fibrosis, but its cellular source and role in regeneration versus fibrosis remain unclear. In this study, we hypothesize that AREG induced in bone marrow-derived CD11c(+) cells is essential for pulmonary fibrosis. Thus, the objectives were to evaluate the importance and role of AREG in pulmonary fibrosis, identify the cellular source of AREG induction, and analyze its regulation of fibroblast function and activation. The results showed that lung AREG expression was significantly induced in bleomycin-induced pulmonary fibrosis. AREG deficiency in knockout mice significantly diminished pulmonary fibrosis. Analysis of AREG expression in major lung cell types revealed induction in fibrotic lungs predominantly occurred in CD11c(+) cells. Moreover, depletion of bone marrow-derived CD11c(+) cells suppressed both induction of lung AREG expression and pulmonary fibrosis. Conversely, adoptive transfer of bone marrow-derived CD11c(+) cells from bleomycin-treated donor mice exacerbated pulmonary fibrosis, but not if the donor cells were made AREG deficient prior to transfer. CD11c(+) cell-conditioned media or coculture stimulated fibroblast proliferation, activation, and myofibroblast differentiation in an AREG-dependent manner. Furthermore, recombinant AREG induced telomerase reverse transcriptase, which appeared to be essential for the proliferative effect. Finally, AREG significantly enhanced fibroblast motility, which was associated with increased expression of α6 integrin. These findings suggested that induced AREG specifically in recruited bone marrow-derived CD11c(+) cells promoted bleomycin-induced pulmonary fibrosis by activation of fibroblast telomerase reverse transcriptase-dependent proliferation, motility, and indirectly, myofibroblast differentiation.

Mesenchymal deficiency of Notch1 attenuates bleomycin-induced pulmonary fibrosis.

Notch signaling pathway is involved in the regulation of cell fate, differentiation, proliferation, and apoptosis in development and disease. Previous studies suggest the importance of Notch1 in myofibroblast differentiation in lung alveogenesis and fibrosis. However, direct in vivo evidence of Notch1-mediated myofibroblast differentiation is lacking. In this study, we examined the effects of conditional mesenchymal-specific deletion of Notch1 on pulmonary fibrosis. Crossing of mice bearing the floxed Notch1 gene with α2(I) collagen enhancer-Cre-ER(T)-bearing mice successfully generated progeny with a conditional knockout (CKO) of Notch1 in collagen I-expressing (mesenchymal) cells on treatment with tamoxifen (Notch1 CKO). Because Notch signaling is known to be activated in the bleomycin model of pulmonary fibrosis, control and Notch1 CKO mice were analyzed for their responses to bleomycin treatment. The results showed significant attenuation of pulmonary fibrosis in CKO relative to control mice, as examined by collagen deposition, myofibroblast differentiation, and histopathology. However, there were no significant differences in inflammatory or immune cell influx between bleomycin-treated CKO and control mouse lungs. Analysis of isolated lung fibroblasts confirmed absence of Notch1 expression in cells from CKO mice, which contained fewer myofibroblasts and significantly diminished collagen I expression relative to those from control mice. These findings revealed an essential role for Notch1-mediated myofibroblast differentiation in the pathogenesis of pulmonary fibrosis.

Reemergence of hedgehog mediates epithelial-mesenchymal crosstalk in pulmonary fibrosis.

Hedgehog signaling plays important roles in cell development and differentiation. In this study, the ability of Sonic Hedgehog (SHH) to induce myofibroblast differentiation was analyzed in isolated human lung fibroblasts, and its in vivo significance was evaluated in rodent bleomycin-induced pulmonary fibrosis. The results showed that SHH could induce myofibroblast differentiation in human lung fibroblasts in a Smo- and Gli1-dependent manner. Gel shift analysis, chromatin immunoprecipitation assay, and site-directed mutagenesis revealed that a Gli1 binding consensus in the α-SMA gene promoter was important for mediating SHH-induced myofibroblast differentiation. Analysis of Hedgehog reemergence in vivo revealed that of all three Hedgehog isoforms, only SHH was significantly induced in bleomycin-injured lung along with Gli1. The induction of SHH was only noted in epithelial cells, and its expression was undetectable in lung fibroblasts or macrophages. transforming growth factor (TGF)-ß induced SHH significantly in cultured alveolar epithelial cells, whereas SHH induced TGF-ß in lung fibroblasts. Pulmonary fibrosis and α-smooth muscle actin (α-SMA) expression were significantly reduced in mice that were Smo deficient only in type I collagen-expressing cells. Thus, the reemergence of SHH in epithelial cells could result in induction of myofibroblast differentiation in a Smo-dependent manner and subsequent Gli1 activation of the α-SMA promoter.

Essential role of stem cell factor-c-Kit signalling pathway in bleomycin-induced pulmonary fibrosis.

Stem cell factor (SCF) and its receptor c-Kit have been implicated in tissue remodelling and fibrosis. Alveolar fibroblasts from patients with diffuse interstitial fibrosis secrete more SCF. However, its precise role remains unclear. In this study the potential role of the SCF-c-Kit axis in pulmonary fibrosis was examined. Fibrosis was induced by intratracheal instillation of bleomycin (BLM), which caused increased SCF levels in plasma, bronchoalveolar lavage fluid (BALF) and lung tissue, as well as increased expression by lung fibroblasts. These changes were accompanied by increased numbers of bone marrow-derived c-Kit(+) cells in the lung, with corresponding depletion in bone marrow. Both recombinant SCF and lung extracts from BLM-treated animals induced bone-marrow cell migration, which was blocked by c-Kit inhibitor. The migrated cells promoted myofibroblast differentiation when co-cultured with fibroblasts, suggesting a paracrine pathogenic role. Interestingly, lung fibroblast cultures contained a subpopulation of cells that expressed functionally active c-Kit, which were significantly greater and more responsive to SCF induction when isolated from fibrotic lungs, including those from patients with idiopathic pulmonary fibrosis (IPF). This c-Kit(+) subpopulation was αSMA-negative and expressed lower levels of collagen I but significantly higher levels of TGFß than c-Kit-negative cells. SCF deficiency achieved by intratracheal treatment with neutralizing anti-SCF antibody or by use of Kitl(Sl)/Kitl(Sl-d) mutant mice in vivo resulted in significant reduction in pulmonary fibrosis. Taken together, the SCF-c-Kit pathway was activated in BLM-injured lung and might play a direct role in pulmonary fibrosis by the recruitment of bone marrow progenitor cells capable of promoting lung myofibroblast differentiation.

Lung bone marrow-derived hematopoietic progenitor cells enhance pulmonary fibrosis.

RATIONALE: Bone marrow (BM)-derived cells have been implicated in pulmonary fibrosis. However, their precise role in pathogenesis is incompletely understood. OBJECTIVES: To elucidate roles of BM-derived cells in bleomycin-induced pulmonary fibrosis, and clarify their potential relationship to lung hematopoietic progenitor cells (LHPCs). METHODS: GFP BM-chimera mice treated with or without bleomycin were used to assess the BM-derived cells. MEASUREMENTS AND MAIN RESULTS: GFP(+) cells in the chimera lung were found to be comprised of two distinct phenotypes: GFP(hi) and GFP(low) cells. The GFP(hi), but not GFP(low), population was significantly increased after bleomycin treatment. Flow-cytometric analysis and quantitative real-time polymerase chain reaction revealed that GFP(hi) cells exhibited phenotypic characteristics of CD11c(+) dendritic cells and macrophages. GFP(hi) cell conditioned media were chemotactic for fibroblasts obtained from fibrotic but not normal lung in vitro. Moreover, adoptive transfer of GFP(hi) cells exacerbated fibrosis in recipient mice, similar to that seen on adoptive transfer of BM-derived CD11c(+) cells from donor bleomycin-treated mice. Next, we evaluated the potential of LHPCs as the source of GFP(hi) cells. Isolation of LHPCs by flow sorting revealed enrichment in cKit(+)/Sca1(-)/Lin(-) cells, most of which were GFP(+) indicating their BM origin. The number of LHPCs increased rapidly after bleomycin treatment. Furthermore, stem cell factor induced LHPC proliferation, whereas granulocyte-macrophage-colony stimulating factor induced differentiation to GFP(hi) cells. CONCLUSIONS: BM-derived LHPCs with a novel phenotype could differentiate into GFP(hi) cells, which enhanced pulmonary fibrosis. Targeting this mobilized LHPCs might represent a novel therapeutic approach in chronic fibrotic lung diseases.

Chronic activation of adrenal Gq signaling induces Cyp11b2 expression in the zona fasciculata and hyperaldosteronism.

Hyperaldosteronism is often associated with inappropriate aldosterone production and aldosterone synthase (Cyp11b2) expression. Normally, Cyp11b2 expression is limited to the adrenal zona glomerulosa (ZG) and regulated by angiotensin II which signals through Gq protein-coupled receptors. As cells migrate inwards, they differentiate into 11ß-hydroxylase-expressing zona fasciculata (ZF) cells lacking Cyp11b2. The mechanism causing ZG-specific aldosterone biosynthesis is still unclear. We investigated the effect of chronic Gq signaling using transgenic mice with a clozapine N-oxide (CNO)-activated human M3 muscarinic receptor (DREADD) coupled to Gq (hM3Dq) that was expressed throughout the adrenal cortex. CNO raised circulating aldosterone in the presence of a high sodium diet with greater response seen in females compared to males. Immunohistochemistry and transcriptomics indicated disrupted zonal Cyp11b2 expression while Wnt signaling remained unchanged. Chronic Gq-DREADD signaling also induced an intra-adrenal RAAS in CNO-treated mice. Chronic Gq signaling disrupted adrenal cortex zonal aldosterone production associated with ZF expression of Cyp11b2.

Telomerase and telomere length in pulmonary fibrosis.

In addition to its expression in stem cells and many cancers, telomerase activity is transiently induced in murine bleomycin (BLM)-induced pulmonary fibrosis with increased levels of telomerase transcriptase (TERT) expression, which is essential for fibrosis. To extend these observations to human chronic fibrotic lung disease, we investigated the expression of telomerase activity in lung fibroblasts from patients with interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF). The results showed that telomerase activity was induced in more than 66% of IPF lung fibroblast samples, in comparison with less than 29% from control samples, some of which were obtained from lung cancer resections. Less than 4% of the human IPF lung fibroblast samples exhibited shortened telomeres, whereas less than 6% of peripheral blood leukocyte samples from patients with IPF or hypersensitivity pneumonitis demonstrated shortened telomeres. Moreover, shortened telomeres in late-generation telomerase RNA component knockout mice did not exert a significant effect on BLM-induced pulmonary fibrosis. In contrast, TERT knockout mice exhibited deficient fibrosis that was independent of telomere length. Finally, TERT expression was up-regulated by a histone deacetylase inhibitor, while the induction of TERT in lung fibroblasts was associated with the binding of acetylated histone H3K9 to the TERT promoter region. These findings indicate that significant telomerase induction was evident in fibroblasts from fibrotic murine lungs and a majority of IPF lung samples, whereas telomere shortening was not a common finding in the human blood and lung fibroblast samples. Notably, the animal studies indicated that the pathogenesis of pulmonary fibrosis was independent of telomere length.

FIZZ2/RELM-ß induction and role in pulmonary fibrosis.

Found in inflammatory zone (FIZZ) 2, also known as resistin-like molecule (RELM)-ß, belongs to a novel cysteine-rich secreted protein family named FIZZ/RELM. Its function is unclear, but a closely related family member, FIZZ1, has profibrotic activities. The human ortholog of rodent FIZZ1 has not been identified, but human FIZZ2 has significant sequence homology to both rodent FIZZ2 (59%) and FIZZ1 (50%). Given the greater homology to rodent FIZZ2, analyzing the role of FIZZ2 in a rodent model of bleomycin-induced pulmonary fibrosis would be of greater potential relevance to human fibrotic lung disease. The results showed that FIZZ2 was highly induced in lungs of rodents with bleomycin-induced pulmonary fibrosis and of human patients with idiopathic pulmonary fibrosis. FIZZ2 expression was induced in rodent and human lung epithelial cells by Th2 cytokines, which was mediated via STAT6 signaling. The FIZZ2 induction in murine lungs was found to be essential for pulmonary fibrosis, as FIZZ2 deficiency significantly suppressed pulmonary fibrosis and associated enhanced extracellular matrix and cytokine gene expression. In vitro analysis indicated that FIZZ2 could stimulate type I collagen and α-smooth muscle actin expression in lung fibroblasts. Furthermore, FIZZ2 was shown to have chemoattractant activity for bone marrow (BM) cells, especially BM-derived CD11c(+) dendritic cells. Notably, lung recruitment of BM-derived cells was impaired in FIZZ2 knockout mice. These findings suggest that FIZZ2 is a Th2-associated multifunctional mediator with potentially important roles in the pathogenesis of fibrotic lung diseases.

Kielin/chordin-like protein, a novel enhancer of BMP signaling, attenuates renal fibrotic disease.

The bone morphogenetic proteins (BMPs) profoundly affect embryonic development, differentiation and disease. BMP signaling is suppressed by cysteine-rich domain proteins, such as chordin, that sequester ligands from the BMP receptor. We describe a novel protein, KCP, with 18 cysteine-rich domains. Unlike chordin, KCP enhances BMP signaling in a paracrine manner. Smad1-dependent transcription and phosphorylated Smad1 (P-Smad1) levels are increased, as KCP binds to BMP7 and enhances binding to the type I receptor. In vivo, Kcp(-/-) mice are viable and fertile. Because BMPs have a pivotal role in renal disease, we examined the phenotype of Kcp(-/-) mice in two different models of renal injury. Kcp(-/-) animals show reduced levels of P-Smad1, are more susceptible to developing renal interstitial fibrosis, are more sensitive to tubular injury and show substantial pathology after recovery. The data indicate an important role for KCP in attenuating the pathology of renal fibrotic disease.

Telomerase activity is required for bleomycin-induced pulmonary fibrosis in mice.

In addition to its well-known expression in the germline and in cells of certain cancers, telomerase activity is induced in lung fibrosis, although its role in this process is unknown. To identify the pathogenetic importance of telomerase in lung fibrosis, we examined the effects of telomerase reverse transcriptase (TERT) deficiency in a murine model of pulmonary injury. TERT-deficient mice showed significantly reduced lung fibrosis following bleomycin (BLM) insult. This was accompanied by a significant reduction in expression of lung alpha-SMA, a marker of myofibroblast differentiation. Furthermore, lung fibroblasts isolated from BLM-treated TERT-deficient mice showed significantly decreased proliferation and increased apoptosis rates compared with cells isolated from control mice. Transplantation of WT BM into TERT-deficient mice restored BLM-induced lung telomerase activity and fibrosis to WT levels. Conversely, transplantation of BM from TERT-deficient mice into WT recipients resulted in reduced telomerase activity and fibrosis. These findings suggest that induction of telomerase in injured lungs may be caused by BM-derived cells, which appear to play an important role in pulmonary fibrosis. Moreover, TERT induction is associated with increased survival of lung fibroblasts, which favors the development of fibrosis instead of injury resolution.

Role of stem cell factor and bone marrow-derived fibroblasts in airway remodeling.

Recent evidence suggests that bone marrow-derived fibroblasts are involved in airway remodeling in asthma, but the role and mechanism of recruitment of these fibroblasts remains unclear. Stem cell factor (SCF), a key factor in the propagation of hematopoietic stem cells, is important in the process of airway remodeling as well. To test the hypothesis that SCF is involved in the recruitment and differentiation of bone marrow-derived progenitor cells, GFP-bone marrow chimeric mice were created. These mice were then sensitized and chronically challenged with cockroach antigen to induce chronic airway disease. Fluorescence microscopy revealed an influx of significant numbers of GFP-expressing fibroblasts in the airways of these mice, which was confirmed by flow cytometric analysis of cells co-expressing both GFP and collagen I. These cells preferentially expressed c-kit, interleukin-31 receptor, and telomerase reverse transcriptase when compared with control lung-derived fibroblasts. Interestingly, SCF stimulated interleukin-31 receptor expression in bone marrow cells, whereas interleukin-31 strongly induced telomerase reverse transcriptase expression in fibroblasts. Treatment with neutralizing antibodies to SCF significantly reduced airway remodeling and suppressed the recruitment of these bone marrow-derived cells to the lung. Thus SCF in conjunction with interleukin-31 may play a significant role in airway remodeling by promoting the recruitment of bone marrow-derived fibroblast precursors into the lung with the capacity to promote lung myofibroblast differentiation.

Notch1 signaling in FIZZ1 induction of myofibroblast differentiation.

Notch1 is an evolutionarily conserved receptor that regulates cell fate, including such events as differentiation, proliferation, and apoptosis. Myofibroblast differentiation is a key feature of lung fibrosis. Found in inflammatory zone 1 (FIZZ1) has direct fibrogenic properties because of its ability to induce myofibroblast differentiation. However, the downstream signaling pathway that mediates FIZZ1 induction of myofibroblast differentiation remains unknown. The objective of this study was to investigate the involvement of Notch signaling in FIZZ1 induction of lung myofibroblast differentiation and thus explore the potential role of Notch1 in pulmonary fibrosis. The results showed that FIZZ1 increased the expression levels of activated intracellular domain of Notch1 (NIC), its ligand Jagged1, and its target gene Hes1, which were associated with elevated alpha-smooth muscle actin expression levels. Fibroblast alpha-smooth muscle actin expression is induced by the overexpression of NIC but is suppressed by the inhibition of NIC. Moreover, lung fibroblasts that were isolated from mice lacking the GDP-4-keto-6-deoxymannose3,5-epimerase-4-reductase enzyme (FX knockout) exhibited significantly reduced responsiveness to FIZZ1, which was reversed by fucose supplementation. In the absence of exogenous fucose, these FX-deficient cells exhibited defective fucosylation, which is required for Notch signaling. These knockout mice also showed impaired lung fibrosis. These findings suggest that Notch1 signaling in response to FIZZ1 may play a significant role in myofibroblast differentiation during lung fibrosis.

Chemogenetic activation of adrenocortical Gq signaling causes hyperaldosteronism and disrupts functional zonation.

The mineralocorticoid aldosterone is produced in the adrenal zona glomerulosa (ZG) under the control of the renin-angiotensin II (AngII) system. Primary aldosteronism (PA) results from renin-independent production of aldosterone and is a common cause of hypertension. PA is caused by dysregulated localization of the enzyme aldosterone synthase (Cyp11b2), which is normally restricted to the ZG. Cyp11b2 transcription and aldosterone production are predominantly regulated by AngII activation of the Gq signaling pathway. Here, we report the generation of transgenic mice with Gq-coupled designer receptors exclusively activated by designer drugs (DREADDs) specifically in the adrenal cortex. We show that adrenal-wide ligand activation of Gq DREADD receptors triggered disorganization of adrenal functional zonation, with induction of Cyp11b2 in glucocorticoid-producing zona fasciculata cells. This result was consistent with increased renin-independent aldosterone production and hypertension. All parameters were reversible following termination of DREADD-mediated Gq signaling. These findings demonstrate that Gq signaling is sufficient for adrenocortical aldosterone production and implicate this pathway in the determination of zone-specific steroid production within the adrenal cortex. This transgenic mouse also provides an inducible and reversible model of hyperaldosteronism to investigate PA therapeutics and the mechanisms leading to the damaging effects of aldosterone on the cardiovascular system.

Animal models of pulmonary fibrosis.

In general, there are two types of animal models: natural and experimental. Because there are no natural models for pulmonary fibrosis, an experimental model that reproduces key aspects of the human disease would be useful for the study of this form of lung disease, the natural history of which is not always known. To date, a variety of animal models have been used to investigate mechanisms of pulmonary and other types of fibrosis, including the intratracheal instillation of bleomycin, fluorescein isothiocyanate, or particulate matter, such as silica and asbestos. This chapter will describe two commonly used techniques, namely bleomycin and silica-induced, which have been developed for the study of pulmonary fibrosis in animal models.

Conditional Knockout of Telomerase Reverse Transcriptase in Mesenchymal Cells Impairs Mouse Pulmonary Fibrosis.

Telomerase is typically expressed in cellular populations capable of extended replication, such as germ cells, tumor cells, and stem cells, but is also induced in tissue injury, repair and fibrosis. Its catalytic component, telomerase reverse transcriptase (TERT) is induced in lung fibroblasts from patients with fibrotic interstitial lung disease and in rodents with bleomycin-induced pulmonary fibrosis. To evaluate the fibroblast specific role of TERT in pulmonary fibrosis, transgenic mice bearing a floxed TERT allele were generated, and then crossed with an inducible collagen α2(I)-Cre mouse line to generate fibroblast specific TERT conditional knockout mice. TERT-specific deficiency in mesenchymal cells caused attenuation of pulmonary fibrosis as manifested by reduced lung hydroxyproline content, type I collagen and α-smooth muscle actin mRNA levels. The TERT-deficient mouse lung fibroblasts displayed decreased cell proliferative capacity and higher susceptibility to induced apoptosis compared with control cells. Additionally TERT deficiency was associated with heightened α-smooth muscle actin expression indicative of myofibroblast differentiation. However the impairment of cell proliferation and increased susceptibility to apoptosis would cause a reduction in the myofibroblast progenitor population necessary to mount a successful myofibroblast-dependent fibrotic response. These findings identified a key role for TERT in fibroblast proliferation and survival essential for pulmonary fibrosis.

Inhibition of key cytokines by tetrathiomolybdate in the bleomycin model of pulmonary fibrosis.

Tetrathiomolybdate is an anticopper drug with a unique mechanism of action. Tetrathiomolybdate complexes copper to protein and itself, rendering the copper unavailable for cellular uptake. It was originally developed for Wilson's disease, and is now being developed as an antiangiogenic agent for the treatment of cancer. Many angiogenic cytokines require normal levels of copper, and lowered copper levels reduce cytokine signaling while cellular copper requirements are met. Cytokines of fibrosis and inflammation may be similarly copper dependent, since tetrathiomolybdate inhibits bleomycin induced pulmonary inflammation and fibrosis. The basis for this inhibition was evaluated here by examination of tetrathiomolybdate effects on cytokines in lung pathophysiologically important in the bleomycin mouse model of pulmonary damage. Results in mice injected endotracheally with bleomycin confirmed that tetrathiomolybdate therapy was effective in reducing fibrosis. This effect was associated with significant inhibition of bleomycin-induced tumor necrosis factor alpha and transforming growth factor beta expression in lung homogenates. These effects were shown to be independent of one another. This indicates that tetrathiomolybdate therapy can be effective even when fibrosis is at a more chronic stage, wherein inflammatory cytokines are playing a diminishing role. The inhibition of tumor necrosis factor alpha suggests that diseases of tumor necrosis factor alpha overexpression are also potential targets of tetrathiomolybdate therapy.

The in vivo fibrotic role of FIZZ1 in pulmonary fibrosis.

FIZZ (found in inflammatory zone) 1, a member of a cysteine-rich secreted protein family, is highly induced in lung allergic inflammation and bleomycin induced lung fibrosis, and primarily expressed by airway and type II alveolar epithelial cells. This novel mediator is known to stimulate α-smooth muscle actin and collagen expression in lung fibroblasts. The objective of this study was to investigate the in vivo effects of FIZZ1 on the development of lung fibrosis by evaluating bleomycin-induced pulmonary fibrosis in FIZZ1 deficient mice. FIZZ1 knockout mice exhibited no detectable abnormality. When these mice were treated with bleomycin they exhibited significantly impaired pulmonary fibrosis relative to wild type mice, along with impaired proinflammatory cytokine/chemokine expression. Deficient lung fibroblast activation was also noted in the FIZZ1 knockout mice. Moreover, recruitment of bone marrow-derived cells to injured lung was deficient in FIZZ1 knockout mice. Interestingly in vitro FIZZ1 was shown to have chemoattractant activity for bone marrow cells, including bone marrow-derived dendritic cells. Finally, overexpression of FIZZ1 exacerbated fibrosis. These findings suggested that FIZZ1 exhibited profibrogenic properties essential for bleomycin induced pulmonary fibrosis, as reflected by its ability to induce myofibroblast differentiation and recruit bone marrow-derived cells.

Gut-enriched Krüppel-like factor interaction with Smad3 inhibits myofibroblast differentiation.

Gut-enriched Krüppel-like factor (GKLF) has been reported to partially inhibit alpha-smooth muscle actin (alpha-SMA) gene transcription by competing for binding to the TGF-beta control element (TCE) with known activators such as Sp1 and other Krüppel-like factors. This incomplete inhibition via the TCE suggests an additional mechanism, which was evaluated in this study. The results showed that an alpha-SMA promoter mutated in the TCE remained susceptible to inhibition by GKLF in rat lung fibroblasts consistent with the existence of an additional TCE-independent mechanism. Since TGF-beta- induced alpha-SMA expression is Smad3-dependent, potential interaction between GKLF and Smad3 was examined as a basis for this additional inhibitory mechanism. Co-immunoprecipitation and yeast two-hybrid assays revealed that GKLF could bind Smad3 through the Smad3 MH2 domain. Electrophoretic mobility shift assays and ChIP assay indicated that this GKLF-Smad3 interaction inhibited Smad3 binding to the Smad3-binding element (SBE) in the alpha-SMA promoter, and the activity of an SBE containing artificial promoter. Further analysis using smad3(-/-) fibroblasts confirmed that the TCE-independent inhibition by GKLF was dependent on Smad3. These data taken together suggest that in addition to inhibition via the TCE, GKLF represses alpha-SMA gene expression by interacting with Smad3 to prevent Smad3 binding to the SBE. It represents the first evidence to directly link GKLF with Smad3, a key intracellular mediator of TGF-beta signaling, which should lead to a clearer understanding of the mechanism of how GKLF regulates TGF-beta-induced gene expression.

Tetrathiomolybdate therapy protects against bleomycin-induced pulmonary fibrosis in mice.

Tetrathiomolybdate (TM), a drug developed for the treatment of Wilson's disease, produces an antiangiogenic effect by reducing systemic copper levels. Several angiogenic cytokines appear to depend on normal levels of copper for activity. In both animal tumor models and in cancer patients, TM therapy has proved effective in inhibiting the growth of tumors. We have hypothesized that the activities of fibrotic and inflammatory cytokines are also subject to modulation by the availability of copper in a manner similar to angiogenic cytokines. As a first step in evaluating whether TM plays a therapeutic role in diseases of inflammation and fibrosis, we studied the effects of TM on a murine model of bleomycin-induced pulmonary fibrosis. Oral TM therapy resulted in dose-dependent reduction in serum ceruloplasmin, a surrogate marker of systemic copper levels. Significant decreases in systemic copper levels were associated with marked reduction in lung fibrosis as determined on the basis of histopathologic findings and a biochemical measure of fibrosis. The protection afforded by TM was also reflected in significantly reduced bleomycin-induced body-weight loss. In the next phase of this work, we will seek to determine the mechanisms by which TM brings about this therapeutic benefit.