Protein tyrosine phosphatase-α amplifies transforming growth factor-ß-dependent profibrotic signaling in lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a progressive, often fatal, fibrosing lung disease for which treatment remains suboptimal. Fibrogenic cytokines, including transforming growth factor-ß (TGF-ß), are central to its pathogenesis. Protein tyrosine phosphatase-α (PTPα) has emerged as a key regulator of fibrogenic signaling in fibroblasts. We have reported that mice globally deficient in PTPα (Ptpra-/-) were protected from experimental pulmonary fibrosis, in part via alterations in TGF-ß signaling. The goal of this study was to determine the lung cell types and mechanisms by which PTPα controls fibrogenic pathways and whether these pathways are relevant to human disease. Immunohistochemical analysis of lungs from patients with IPF revealed that PTPα was highly expressed by mesenchymal cells in fibroblastic foci and by airway and alveolar epithelial cells. To determine whether PTPα promotes profibrotic signaling pathways in lung fibroblasts and/or epithelial cells, we generated mice with conditional (floxed) Ptpra alleles (Ptpraf/f). These mice were crossed with Dermo1-Cre or with Sftpc-CreERT2 mice to delete Ptpra in mesenchymal cells and alveolar type II cells, respectively. Dermo1-Cre/Ptpraf/f mice were protected from bleomycin-induced pulmonary fibrosis, whereas Sftpc-CreERT2/Ptpraf/f mice developed pulmonary fibrosis equivalent to controls. Both canonical and noncanonical TGF-ß signaling and downstream TGF-ß-induced fibrogenic responses were attenuated in isolated Ptpra-/- compared with wild-type fibroblasts. Furthermore, TGF-ß-induced tyrosine phosphorylation of TGF-ß type II receptor and of PTPα were attenuated in Ptpra-/- compared with wild-type fibroblasts. The phenotype of cells genetically deficient in PTPα was recapitulated with the use of a Src inhibitor. These findings suggest that PTPα amplifies profibrotic TGF-ß-dependent pathway signaling in lung fibroblasts.

Osteogenesis requires FAK-dependent collagen synthesis by fibroblasts and osteoblasts.

Focal adhesion kinase (FAK) is critical in adhesion-dependent signaling, but its role in osteogenesis in vivo is ill defined. We deleted Fak in fibroblasts and osteoblasts in Floxed-Fak mice bred with those expressing Cre-recombinase driven by 3.6-kb α1(I)-collagen promoter. Compared with wild-type (WT), conditional FAK-knockout (CFKO) mice were shorter (2-fold; P < 0.0001) and had crooked, shorter tails (50%; P < 0.0001). Microcomputed tomography analysis showed reduced bone volume (4-fold in tails; P < 0.0001; 2-fold in mandibles; P < 0.0001), whereas bone surface area/bone volume increased (3-fold in tails; P < 0.0001; 2.5-fold in mandibles; P < 0.001). Collagen density and fiber alignment in periodontal ligament were reduced by 4-fold (P < 0.0001) and 30% (P < 0.05), respectively, in CFKO mice. In cultured CFKO osteoblasts, mineralization at d 7 and mineralizing colony-forming units at d 21 were 30% (P < 0.0001) and >3-fold less than WT, respectively. Disruptions of FAK function in osteoblasts by conditional knockout, siRNA-knockdown, or FAK inhibitor reduced mRNA and protein expression of Runx2 (>30%), Osterix (>25%), and collagen-1 (2-fold). Collagen synthesis was abrogated in WT osteoblasts with Runx2 knockdown and in Fak-null fibroblasts transfected with an FAK kinase domain mutant or a kinase-impaired mutant (Y397F). These data indicate that FAK regulates osteogenesis through transcription factors that regulate collagen synthesis.-Rajshankar, D., Wang, Y., McCulloch, C. A. Osteogenesis requires FAK-dependent collagen synthesis by fibroblasts and osteoblasts.

IL-1ß enhances cell adhesion to degraded fibronectin.

IL-1ß is a prominent proinflammatory cytokine that mediates degradation of extracellular matrix proteins through increased expression of matrix metalloproteinases, which involves a signaling pathway in adherent cells that is restricted by focal adhesions. Currently, the mechanism by which IL-1ß affects cell adhesion to matrix proteins is not defined, and it is not known whether degraded matrix proteins affect IL-1ß signaling. We examined adhesion-related IL-1ß signaling in fibroblasts attaching to native or MMP3-degraded fibronectin. IL-1ß increased cell attachment, resistance to shear force and the numbers of focal adhesions containing activated ß(1) integrins. IL-1ß-enhanced attachment required FAK, kindlins 1/2, and talin. MMP3-degraded fibronectin-inhibited IL-1ß-enhanced cell adhesion and promoted spontaneous ERK activation that was independent of IL-1ß treatment. We conclude that IL-1ß enhances the adhesion of anchorage-dependent cells to MMP3-degraded fibronectin, which, in turn, is associated with deregulated cellular responses to IL-1ß. These data point to a novel role of IL-1ß as a proadhesive signaling molecule in inflammation that employs kindlins and talin to regulate adhesion.