Hongfeng Yu

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.

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.

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-β Induction and Role in Pulmonary Fibrosis

Found in inflammatory zone (FIZZ) 2, also known as resistin-like molecule (RELM)-β, 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.

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.

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.

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.