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Idiopathic pulmonary fibrosis (IPF) is a progressive, often fatal, fibrosing lung disease for which treatment remains suboptimal. Fibrogenic cytokines, including transforming growth factor-β (TGF-β), 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-β 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 ( Ptpra f/f ). These mice were crossed with Dermo1 -Cre or with Sftpc -CreER T2 mice to delete Ptpra in mesenchymal cells and alveolar type II cells, respectively. Dermo1 -Cre/ Ptpra f/f mice were protected from bleomycin-induced pulmonary fibrosis, whereas Sftpc- CreER T2 /Ptpra f/f mice developed pulmonary fibrosis equivalent to controls. Both canonical and noncanonical TGF-β signaling and downstream TGF-β-induced fibrogenic responses were attenuated in isolated Ptpra -/- compared with wild-type fibroblasts. Furthermore, TGF-β-induced tyrosine phosphorylation of TGF-β 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-β-dependent pathway signaling in lung fibroblasts.

Focal adhesion kinase (FAK) is critical for collagen expression but its regulation of collagen remodeling is not defined. We examined the role of FAK in the degradation and reorganization of fibrillar collagen. Compared with wild-type (WT) mouse embryonic fibroblasts, FAK null (FAK -/- ) fibroblasts generated twofold (p < .0001) higher levels of ¾ collagen I fragment and expressed up to fivefold more membrane-type matrix metalloproteinase (MMP). When plated on stiff collagen substrates, compared with WT, FAK -/- cells were smaller (threefold reduced cell surface area; p < .0001) and produced fivefold fewer cell extensions (p < .0001) that were 40% shorter (p < .001). When cultured on soft collagen gels (stiffness of ~100 Pa) for 6-48 hr, cell spreading and cell extension formation were reduced by greater than twofold (p < .05 and p < .0001, respectively) while collagen compaction and alignment were reduced by approximately 30% (p < .0001) in FAK -/- cells. Similar results were found after treatment with PF573228, a FAK inhibitor. Reconstitution of FAK -/- cells with FAK mutants showed that compared with WT, cell extension formation was reduced twofold (p < .0001) in the absence of the kinase domain and sixfold (p < .0001) with a Y397F mutant. Enhanced collagen degradation was exhibited by the mutants (~threefold increase; p < .0001 of ¾ collagen fragments without kinase domain or Y397F mutant; p < .01). Compared with FAK +/+ cells, matrices produced by FAK -/- cells generated higher levels of β 1 integrin activation (p < 0.05), extracellular-signal-regulated kinase (ERK) phosphorylation, and production of ¾ collagen I fragment by human gingival fibroblasts. Collectively these data indicate that (a) the kinase activity of FAK enhances collagen remodeling by tractional forces but inhibits collagen degradation by MMPs; (b) FAK influences the biological activity of fibroblast-secreted extracellular matrices, which in turn impacts β1 integrin and ERK signaling, and collagen degradation., (© 2019 Wiley Periodicals, Inc.)

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., (© FASEB.)

The application of nanotechnology for drug targeting underlines the importance of controlling the kinetics and cellular sites of delivery for optimal therapeutic outcomes. Here we examined the effect of particle size on internalization and degradation of surface-bound fibronectin by fibroblasts using polystyrene nanoparticles (NPs; 51 nm) and microparticles (MPs; 1 μm). Fibronectin was strongly bound by NPs and MPs as assessed by immuno-dot blot analysis (5.1 ± 0.4 × 10(- 5)pg fibronectin per μm(2) of NP surface; 4.2 ± ± 0.3 × 10(-5)pg fibronectin per μm(2) of MP surface; p>0.2). We estimated that ~193 fibronectin molecules bound to a MP compared with 0.6 fibronectin molecules per NP, indicating that ~40% of nanoparticles were not bound by fibronectin. One hour after incubation, fibronectin-coated NPs and MPs were rapidly internalized by Rat-2 fibroblasts. MPs and NPs were engulfed partly by receptor-mediated endocytosis as indicated by decreased uptake when incubated at 4°C, or by depletion of ATP with sodium azide. Pulse-chase experiments showed minimal exocytosis of NPs and MPs. Internalization of NPs and MPs was inhibited by jasplakinolide, whereas internalization of MPs but not NPs was inhibited by latrunculin B and by integrin-blocking antibodies. Extraction of plasma membrane cholesterol with methyl β-cyclodextrin inhibited internalization of fibronectin-coated NPs but not MPs. Biotinylated fibronectin internalized by cells was extensively degraded on MPs but not NPs. Particle size affects actin and clathrin-dependent internalization mechanisms leading to fibronectin degradation on MPs but not NPs. Thus either prolonged, controlled release or an immediate delivery of drugs can be achieved by adjusting the particle size along with matrix proteins such as FN., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Interleukin-1 (IL-1) signaling in fibroblasts is mediated through focal adhesions, organelles that are enriched with adaptor and cytoskeletal proteins that regulate signal transduction. We examined interactions of the focal adhesion kinase (FAK) with protein-tyrosine phosphatase-α (PTP-α) in IL-1 signaling. In wild type and FAK knock-out mouse embryonic fibroblasts, we found by immunoblotting, immunoprecipitation, immunostaining, and gene silencing that FAK is required for IL-1-mediated sequestration of PTPα to focal adhesions. Immunoprecipitation and pulldown assays of purified proteins demonstrated a direct interaction between FAK and PTPα, which was dependent on the FAT domain of FAK and by an intact membrane-proximal phosphatase domain of PTPα. Recruitment of PTPα to focal adhesions, IL-1-induced Ca(2+) release from the endoplasmic reticulum, ERK activation, and IL-6, MMP-3, and MMP-9 expression were all blocked in FAK knock-out fibroblasts. These processes were restored in FAK knock-out cells transfected with wild type FAK, FAT domain, and FRNK. Our data indicate that IL-1-induced signaling through focal adhesions involves interactions between the FAT domain of FAK and PTPα., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Adhesion molecules expressed by periodontal connective tissue cells are involved in cell migration, matrix remodeling and inflammatory responses to infection. Currently, the processes by which the biologic activity of these molecules are appropriately regulated in time and space to preserve tissue homeostasis, and to control inflammatory responses and tissue regeneration, are not defined. As cell adhesions are heterogeneous, dynamic, contain a complex group of interacting molecules and are strongly influenced by the type of substrate to which they adhere, we focus on how cell adhesions in periodontal connective tissues contribute to information generation and processing that regulate periodontal structure and function. We also consider how proteomic methods can be applied to discover novel cell-adhesion proteins that could potentially contribute to the form and function of periodontal tissues., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Collagen degradation by phagocytosis is essential for physiological collagen turnover and connective tissue homeostasis. The rate limiting step of phagocytosis is the binding of specific adhesion receptors, which include the integrins and discoidin domain receptors (DDR), to fibrillar collagen. While previous data suggest that these two receptors interact, the functional nature of these interactions is not defined. In mouse and human fibroblasts we examined the effects of DDR1 knockdown and over-expression on β1 integrin subunit function. DDR1 expression levels were positively associated with enhanced contraction of floating and attached collagen gels, increased collagen binding and increased collagen remodeling. In DDR1 over-expressing cells compared with control cells, there were increased numbers, area and length of focal adhesions immunostained for talin, paxillin, vinculin and activated β1 integrin. After treatment with the integrin-cleaving protease jararhagin, in comparison to controls, DDR1 over-expressing cells exhibited increased β1 integrin cleavage at the cell membrane, indicating that DDR1 over-expression affected the access and susceptibility of cell-surface β1 integrin to the protease. DDR1 over-expression was associated with increased glycosylation of the β1 integrin subunit, which when blocked by deoxymannojirimycin, reduced collagen binding. Collectively these data indicate that DDR1 regulates β1 integrin interactions with fibrillar collagen, which positively impacts the binding step of collagen phagocytosis and collagen remodeling.

IL-1β contributes to connective tissue destruction in part by up-regulating stromelysin-1 (MMP-3), which in fibroblasts is a focal adhesion-dependent process. Protein tyrosine phosphatase-α (PTPα) is enriched in and regulates the formation of focal adhesions, but the role of PTPα in connective tissue destruction is not defined. We first examined destruction of periodontal connective tissues in adult PTPα(+/+) and PTPα(-/-) mice subjected to ligature-induced periodontitis, which increases the levels of multiple cytokines, including IL-1β. Three weeks after ligation, maxillae were processed for morphometry, micro-computed tomography and histomorphometry. Compared with unligated controls, there was ∼1.5-3 times greater bone loss as well as 3-fold reduction of the thickness of the gingival lamina propria and 20-fold reduction of the amount of collagen fibers in WT than PTPα(-/-) mice. Immunohistochemical staining of periodontal tissue showed elevated expression of MMP-3 at ligated sites. Second, to examine mechanisms by which PTPα may regulate matrix degradation, human MMP arrays were used to screen conditioned media from human gingival fibroblasts treated with vehicle, IL-1β or TNFα. Although MMP-3 was upregulated by both cytokines, only IL-1β stimulated ERK activation in human gingival fibroblasts plated on fibronectin. TIRF microscopy and immunoblotting analyses of cells depleted of PTPα activity with the use of various mutated constructs or with siRNA or PTPα(KO) and matched wild type fibroblasts were plated on fibronectin to enable focal adhesion formation and stimulated with IL-1β. These data showed that the catalytic and adaptor functions of PTPα were required for IL-1β-induced focal adhesion formation, ERK activation and MMP-3 release. We conclude that inflammation-induced connective tissue degradation involving fibroblasts requires functionally active PTPα and in part is mediated by IL-1β signaling through focal adhesions.

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

Interleukin-1 (IL-1) induces extracellular matrix degradation as a result of increased expression of matrix metalloproteinases (MMPs). We examined adhesion-restricted signaling pathways that enable IL-1-induced MMP release in human gingival and murine fibroblasts. Of the seven MMPs and three tissue inhibitors of MMPs screened, IL-1 enhanced release only of MMP3 when cells formed focal adhesions. Inhibition of protein-tyrosine phosphatases (PTPs), which are enriched in focal adhesions, blocked IL-1-induced MMP3 release. Accordingly, in contrast to wild-type cells, fibroblasts null for PTPalpha did not exhibit IL-1-induced MMP3 release. IL-1 treatment enhanced the recruitment of SHP-2 and PTPalpha to focal adhesions and the association of PTPalpha with SHP-2. Pulldown assays confirmed a direct interaction between PTPalpha and SHP-2, which was dependent on the intact, membrane-proximal phosphatase domain of PTPalpha. Interactions between SHP-2 and PTPalpha, recruitment of SHP-2 to focal adhesions, IL-1-induced ERK activation, and MMP3 expression were all blocked by point mutations in the phosphatase domains of PTPalpha. These data indicate that IL-1-induced signaling through focal adhesions leading to MMP3 release and interactions between SHP-2 and PTPalpha are dependent on the integrity of the catalytic domains of PTPalpha.

Calcium (Ca2+) signaling by the pro-inflammatory cytokine interleukin-1 (IL-1) is dependent on focal adhesions, which contain diverse structural and signaling proteins including protein phosphatases. We examined here the role of protein-tyrosine phosphatase (PTP) alpha in regulating IL-1-induced Ca2+ signaling in fibroblasts. IL-1 promoted recruitment of PTPalpha to focal adhesions and endoplasmic reticulum (ER) fractions, as well as tyrosine phosphorylation of the ER Ca2+ release channel IP3R. In response to IL-1, catalytically active PTPalpha was required for Ca2+ release from the ER, Src-dependent phosphorylation of IP3R1 and accumulation of IP3R1 in focal adhesions. In pulldown assays and immunoprecipitations PTPalpha was required for the association of PTPalpha with IP3R1 and c-Src, and this association was increased by IL-1. Collectively, these data indicate that PTPalpha acts as an adaptor to mediate functional links between focal adhesions and the ER that enable IL-1-induced Ca2+ signaling.

Periodontal ligament width is precisely maintained throughout the lifetime of adult mammals but the biological mechanisms that inhibit ingrowth of bone into this soft connective tissue are unknown. As bone morphogenic proteins strongly stimulate osteogenesis and can induce ectopic bone formation in vivo, we tested the hypothesis that topical application of this powerful osteogenic agent will overwhelm the osteogenic inhibitory mechanisms of periodontal ligament cells and induce ankylosis. Wounds through the alveolar bone and periodontal ligament were created in 45 male Wistar rats. Defects were filled with either a collagen implant or collagen plus bone morphogenic protein (BMP-7), or were left unfilled (controls). Three animals per time period were killed on days 2, 5, 10, 21 and 60 after surgery for each wound type. Cellular proliferation and clonal growth in periodontal tissues were assessed by 3H-thymidine labeling 1 h before death, followed by radioautography. Cellular differentiation of soft and mineralizing connective tissue cell populations was determined by immunohistochemical staining of alpha-smooth muscle actin, osteopontin and bone sialoprotein. In regenerating periodontium, BMP-7 induced abundant bone formation by 21 days (2.5-fold greater than controls or collagen implant only; P<0.001), but by day 60 the volume of the newly formed bone had returned to baseline levels and was similar for all groups. Independent of the type of treatment, periodontal ligament width was unchanged throughout the experimental period (P>0.05). Animals treated with BMP-7 implants showed greatly increased cellular proliferation in the periodontal ligament adjacent to the wound site and in the regenerating alveolar bone at days 5 and 10 after wounding compared to the other treatment groups (P<0.005). Animals in the BMP-7 group exhibited similar spatial and temporal staining patterns for alpha-smooth muscle actin, osteopontin and bone sialoprotein as controls. Collectively, these data show that BMP-7 promoted the proliferation of precursor cells in the periodontal ligament but did not induce osteogenic differentiation in this compartment. Consequently a powerful osteogenic stimulus like BMP-7 cannot significantly perturb the mechanisms that regulate periodontal ligament width and maintain periodontal homeostasis.