Amelia K. Scaffidi

Fibroblasts Isolated from Normal Lungs and Those with Idiopathic Pulmonary Fibrosis Differ in Interleukin-6/gp130-Mediated Cell Signaling and Proliferation

Interleukin (IL)-6 and IL-11 are elevated in a variety of lung conditions and may impact on repair mechanisms in chronic inflammatory disorders. However, the mechanisms by which these cytokines influence fibroblast proliferation in normal and disease states have not been previously addressed. We examined the effect of these cytokines on proliferation and cell-cycle kinetics of primary human lung fibroblasts obtained from normal patients and patients with idiopathic pulmonary fibrosis (IPF). IL-6 inhibited the proliferation of normal fibroblasts due to the sustained phosphorylation of STAT-3 and production of the cyclin-dependent kinase inhibitor p19(INK4D). In contrast IL-6 was mitogenic for IPF fibroblasts due to the sustained activation of MAPK, which in turn inhibited the production of p27(Kip1), allowing activation of cyclin D(1) and hyperphosphorylation of retinoblastoma protein. IL-11 was mitogenic for both normal and IPF fibroblasts. These results provide strong evidence for a fundamental abnormality in a cytokine-signaling pathway, as opposed to alterations in cytokine production, in the pathogenesis of IPF.

αvβ3 Integrin Interacts with the Transforming Growth Factor β (TGFβ) Type II Receptor to Potentiate the Proliferative Effects of TGFβ1 in Living Human Lung Fibroblasts

The αvβ3 integrin is known to cooperate with receptor tyrosine kinases to enhance cellular responses. To determine whether αvβ3 regulates transforming growth factor β (TGFβ) 1-induced responses, we investigated the interaction between αvβ3 and TGFβ type II receptor (TGFβIIR) in primary human lung fibroblasts. We report that TGFβ1 up-regulates cell surface and mRNA expression of αvβ3 in a time- and dose-dependent manner. Co-immunoprecipitation and confocal microscopy showed that TGFβRII associates and clusters with αvβ3, following TGFβ1 exposure. This association was not observed with αvβ5 or α5β1. We also used a novel molecular proximity assay, bioluminescence resonance energy transfer (BRET), to quantify this dynamic interaction in living cells. TGFβ1 stimulation resulted in a BRET signal within 5 min, whereas tenascin, which binds αvβ3, did not induce a substantial BRET signal. Co-exposure to tenascin and TGFβ1 produced no further increases in BRET than TGFβ1 alone. Cyclin D1 was rapidly induced in cells co-exposed to TGFβ1 and tenascin, and as a consequence proliferation induced by TGFβ1 was dramatically enhanced in cells co-exposed to tenascin or vitronectin. Cholesterol depletion inhibited the interaction between TGFβRII and αvβ3 and abrogated the proliferative effect. The cyclic RGD peptide, GpenGRGDSPCA, which blocks αvβ3, also abolished the synergistic proliferative effect seen. These results indicate a new interaction partner for the αvβ3 integrin, the TGFβIIR, in which TGFβ1-induced responses are potentiated in the presence αvβ3 ligands. Our data provide a novel mechanism by which TGFβ1 may contribute to abnormal wound healing and tissue fibrosis.

Macrophage recognition and phagocytosis of apoptotic fibroblasts is critically dependent on fibroblast-derived thrombospondin 1 and CD36

The induction of fibroblast apoptosis and their clearance by phagocytes is essential for normal wound healing and prevention of scarring. However, little is known about the clearance of apoptotic fibroblasts and whether apoptotic cells are active participants in the recruitment and activation of phagocytes. In this study, we provide the first evidence that apoptotic fibroblasts actively release increased amounts of thrombospondin (TSP1) to actively recruit macrophages. Expression of TSP1 and its receptor CD36 was increased on the surface of apoptotic fibroblasts. By chemical cross-linking and immunoprecipitation we show that TSP1 and CD36 were directly associated. This was confirmed by confocal microscopy. Blockade of either CD36 or TSP1 on apoptotic fibroblasts inhibited phagocytosis. Blockade of alpha v beta 3 integrins as well as CD36 and TSP1 on macrophages inhibited phagocytosis. In contrast, phosphatidylserine or lectins were not involved. These findings suggest that apoptotic fibroblasts release TSP1 as a signal to recruit macrophages while the up-regulated expression of the CD36/TSP1 complex on their cell surface may form a ligand bridging the fibroblast to a complex consisting of alpha v beta 3/CD36/TSP1 on macrophages. These results establish fundamental mechanisms for the clearance of apoptotic fibroblasts and may provide insights into the processes involved in normal wound repair.

Myotube Formation is Delayed but not Prevented in MyoD-deficient Skeletal Muscle: Studies in Regenerating Whole Muscle Grafts of Adult Mice:

We compared the time course of myogenic events in vivo in regenerating whole muscle grafts in MyoD(−/−) and control BALB/c adult mice using immunohistochemistry and electron microscopy. Immunohistochemistry with antibodies to desmin and myosin revealed a striking delay by about 3 days in the formation of myotubes in MyoD(−/−) autografts compared with BALB/c mice. However, myotube formation was not prevented, and autografts in both strains appeared similar by 8 days. Electron microscopy confirmed myotube formation in 8- but not 5-day MyoD(−/−) grafts. This pattern was not influenced by cross-transplantation experiments between strains examined at 5 days. Antibodies to proliferating cell nuclear antigen demonstrated an elevated level of replication by MyoD(−/−) myoblasts in autografts, and replication was sustained for about 3 days compared with controls. These data indicate that the delay in the onset of differentiation and hence fusion is related to extended proliferation of the MyoD(−/−) myoblasts. Overall, although muscle regeneration was delayed it was not impaired in MyoD(−/−) mice in this model.

Comparison of the morphological and biochemical changes in normal human lung fibroblasts and fibroblasts derived from lungs of patients with idiopathic pulmonary fibrosis during FasL-induced apoptosis.

It is increasingly recognized that the morphological changes of apoptosis vary between cell types. This heterogeneity reflects the wide range of cellular proteins and enzymes involved in apoptotic pathways. Fibroblast apoptosis is crucial to the regression of scars and the restitution of healthy tissue during wound repair and may be aberrant in diseases such as idiopathic pulmonary fibrosis (IPF). The biochemical and morphological changes characterizing fibroblast apoptosis are unknown and may provide insights into the specific enzymatic mediators activated in these cells. This study aimed to examine the morphological changes of fibroblast apoptosis in both primary normal lung fibroblasts (normal‐Fb) and fibroblasts obtained from patients with idiopathic pulmonary fibrosis (IPF‐Fb) and to correlate these changes with conventional biochemical markers. Transmission electron microscopy (TEM) and video time‐lapse microscopy demonstrated no difference in the duration of fibroblast apoptosis in response to FasL (6 ± 0.3 h in normal‐Fb and 6.4 ± 0.2 h in IPF‐Fb). However, IPF‐Fb were more resistant to FasL‐induced apoptosis compared with normal‐Fb. Although the majority of morphological changes of normal‐Fb and IPF‐Fb were similar, the formation of filopodia and condensation of the cytoskeletal bundles in IPF‐Fb, and more prominent vacuolation in normal‐Fb, were the significant differences between these cell subtypes. Loss of the mitochondrial membrane potential occurred prior to caspase‐3 activation, while phosphatidylserine expression, cytokeratin‐18 cleavage, and DNA fragmentation commenced after caspase‐3 activation. These observations not only suggest that specific enzymatic effectors may be preferentially activated during fibroblast apoptosis, but also provide potential insights into the pathogenesis of IPF. Copyright

Transforming Growth Factor β1 Induces αvβ3 Integrin Expression in Human Lung Fibroblasts via a β3 Integrin-, c-Src-, and p38 MAPK-dependent Pathway

In response to transforming growth factor β1 (TGFβ) stimulation, fibroblasts modify their integrin repertoire and adhesive capabilities to certain extracellular matrix proteins. Although TGFβ has been shown to increase the expression of specific αv integrins, the mechanisms underlying this are unknown. In this study we demonstrate that TGFβ1 increased both β3 integrin subunit mRNA and protein levels as well as surface expression of αvβ3 in human lung fibroblasts. TGFβ1-induced αvβ3 expression was strongly adhesion-dependent and associated with increased focal adhesion kinase and c-Src kinase phosphorylation. Inhibition of β3 integrin activation by the Arg-Gly-Asp tripeptide motif-specific disintegrin echistatin or αvβ3 blocking antibody prevented the increase in β3 but not β5 integrin expression. In addition, echistatin inhibited TGFβ1-induced p38 MAPK but not Smad3 activation. Furthermore, inhibition of the Src family kinases, but not focal adhesion kinase, completely abrogated TGFβ1-induced expression of αvβ3 and p38 MAPK phosphorylation but not β5 integrin expression and Smad3 activation. The TGFβ1-induced αvβ3 expression was blocked by pharmacologic and genetic inhibition of p38 MAPK- but not Smad2/3-, Sp1-, ERK-, phosphatidylinositol 3-kinase, and NF-κB-dependent pathways. Our results demonstrate that TGFβ1 induces αvβ3 integrin expression via a β3 integrin-, c-Src-, and p38 MAPK-dependent pathway. These data identify a novel mechanism for TGFβ1 signaling in human lung fibroblasts by which they may contribute to normal and pathological wound healing.

Oncostatin M stimulates proliferation, induces collagen production and inhibits apoptosis of human lung fibroblasts

Oncostatin M (OSM), a member of the interleukin‐6 (IL‐6) cytokine family, acts on a variety of cells and elicits diversified biological responses, suggesting potential roles in the regulation of cell survival, differentiation and proliferation. We have examined the effect of OSM on the regulation of human lung fibroblast proliferation, collagen production and spontaneous apoptosis. The proliferative effects of OSM (0.5 – 100 ng ml−1) were assessed using a MTS assay as well as [3H]‐thymidine incorporation and cell counts at 24 and 48 h. Hydroxyproline was measured as an index of procollagen production by high pressure liquid chromotography (HPLC). Apoptosis was determined by annexin staining. OSM enhanced the mitotic activity of lung fibroblasts in a time and dose dependent manner. Maximum proliferation of 57% above control was observed after incubation for 48 h with 2 ng ml−1 OSM (P<0.05). Incubation with the mitogen activated protein kinase (MAPK) kinase inhibitor, PD98059 or the tyrosine kinase inhibitor, genestein both significantly reduced the mitogenic effect of OSM (P<0.05). In contrast, proliferation in response to OSM was not regulated by induction of cyclo‐oxygenase and subsequent prostaglandin E2 (PGE2) release or by IL‐6. OSM also stimulated fibroblasts to synthesize pro‐collagen by a maximum of 35% above control levels after 48 h (P<0.05). OSM significantly inhibited the spontaneous apoptosis of fibroblasts at 24 and 48 h. These results provide evidence that OSM has pro‐fibrotic properties and suggest that it may play a role in normal lung wound repair and fibrosis.

αvβ3 integrin interacts with the TGFβ type II receptor to potentiate the proliferative effects of TGFβ1 in living human lung fibroblasts

The αvβ3 integrin is known to cooperate with receptor tyrosine kinases to enhance cellular responses. To determine whether αvβ3 regulates transforming growth factor β (TGFβ) 1-induced responses, we investigated the interaction between αvβ3 and TGFβ type II receptor (TGFβIIR) in primary human lung fibroblasts. We report that TGFβ1 up-regulates cell surface and mRNA expression of αvβ3 in a time- and dose-dependent manner. Co-immunoprecipitation and confocal microscopy showed that TGFβRII associates and clusters with αvβ3, following TGFβ1 exposure. This association was not observed with αvβ5 or α5β1. We also used a novel molecular proximity assay, bioluminescence resonance energy transfer (BRET), to quantify this dynamic interaction in living cells. TGFβ1 stimulation resulted in a BRET signal within 5 min, whereas tenascin, which binds αvβ3, did not induce a substantial BRET signal. Co-exposure to tenascin and TGFβ1 produced no further increases in BRET than TGFβ1 alone. Cyclin D1 was rapidly induced in cells co-exposed to TGFβ1 and tenascin, and as a consequence proliferation induced by TGFβ1 was dramatically enhanced in cells co-exposed to tenascin or vitronectin. Cholesterol depletion inhibited the interaction between TGFβRII and αvβ3 and abrogated the proliferative effect. The cyclic RGD peptide, GpenGRGDSPCA, which blocks αvβ3, also abolished the synergistic proliferative effect seen. These results indicate a new interaction partner for the αvβ3 integrin, the TGFβIIR, in which TGFβ1-induced responses are potentiated in the presence αvβ3 ligands. Our data provide a novel mechanism by which TGFβ1 may contribute to abnormal wound healing and tissue fibrosis.

TGFβ1 induces αvβ3 integrin expression in human lung fibroblasts via a β3 integrin, c-Src and p38MAPK dependent pathway

In response to transforming growth factor β1 (TGFβ) stimulation, fibroblasts modify their integrin repertoire and adhesive capabilities to certain extracellular matrix proteins. Although TGFβ has been shown to increase the expression of specific αv integrins, the mechanisms underlying this are unknown. In this study we demonstrate that TGFβ1 increased both β3 integrin subunit mRNA and protein levels as well as surface expression of αvβ3 in human lung fibroblasts. TGFβ1-induced αvβ3 expression was strongly adhesion-dependent and associated with increased focal adhesion kinase and c-Src kinase phosphorylation. Inhibition of β3 integrin activation by the Arg-Gly-Asp tripeptide motif-specific disintegrin echistatin or αvβ3 blocking antibody prevented the increase in β3 but not β5 integrin expression. In addition, echistatin inhibited TGFβ1-induced p38 MAPK but not Smad3 activation. Furthermore, inhibition of the Src family kinases, but not focal adhesion kinase, completely abrogated TGFβ1-induced expression of αvβ3 and p38 MAPK phosphorylation but not β5 integrin expression and Smad3 activation. The TGFβ1-induced αvβ3 expression was blocked by pharmacologic and genetic inhibition of p38 MAPK- but not Smad2/3-, Sp1-, ERK-, phosphatidylinositol 3-kinase, and NF-κB-dependent pathways. Our results demonstrate that TGFβ1 induces αvβ3 integrin expression via a β3 integrin-, c-Src-, and p38 MAPK-dependent pathway. These data identify a novel mechanism for TGFβ1 signaling in human lung fibroblasts by which they may contribute to normal and pathological wound healing.

Assessment of p.Phe508del-CFTR functional restoration in pediatric primary cystic fibrosis airway epithelial cells

Background Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene can reduce function of the CFTR ion channel activity and impair cellular chloride secretion. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC. Methods Pediatric pAECs derived from children with CF (pAECCF) were established and expanded as monolayer cultures, before seeding into 96-well plates for the halide assay. Cells were then transduced with an adenoviral construct containing yellow fluorescent protein (eYFP) reporter gene, alone or in combination with either wild-type CFTR (WT-CFTR) or p.Phe508del CFTR. Four days post transduction, cells were stimulated with forskolin and genistein, and assessed for quenching of the eYFP signal following injection of iodide solution into the assay media. Results Data showed that pAECCF can express eYFP at high efficiency following transduction with the eYFP construct. The halide assay was able to discriminate functional restoration of CFTR in pAECCF treated with either WT-CFTR construct or the positive controls syntaxin 8 and B-cell receptor-associated protein 31 shRNAs. Significance The current study demonstrates that the halide assay can be adapted for pediatric pAECCF to evaluate restoration of CFTR function. With the ongoing development of small molecules to modulate the folding and/or activity of various mutated CFTR proteins, this halide assay presents a small-scale personalized screening platform that could assess therapeutic potential of molecules across a broad range of CFTR mutations.