Lysyl oxidase like-2 (LOXL2) was found to be present extracellularly in primary human gingival fibroblast cells. This project has been primarily focused on investigating our hypothesis that LOXL2 may play a critical role in regulating cell proliferation and collagen accumulation in primary human gingival fibroblast cells, which may contribute to the development of fibrotic changes in human gingival tissue. LOXL2 shRNA lentivirus reduced the LOXL2 mRNA and protein expression by 90 – 95%. Knockdown of LOXL2 or inhibition of LOXL2 enzymatic activity strongly inhibited both basal and CCN2/CTGF-stimulated collagen accumulation (p<0.05). Proliferation assays demonstrated a marked decrease in cell proliferation in both the short and long term in LOXL2 shRNA knockdown cells with minimal or no stimulation of cell apoptosis. Pharmacologic inhibition of LOXL2 enzyme activity reduced basal and CCN2/CTGF-stimulated cell proliferation (40% and 50%) in short term cultures. Furthermore, there was 15-20% inhibition seen in long term assays. Recombinant active LOXL2 significantly increased collagen accumulation and cell proliferation (p<0.05). Thereby, our investigation in vitro by loss and gain of function experiments confirmed that LOXL2 is critically required for both gingival fibroblast proliferation and for collagen accumulation in the presence or absence of CCN2/CTGF. LOXL2 stimulation is critical for both proliferation and collagen accumulation in primary human gingival fibroblasts. Lastly, we found that the presence of LOXL2 extracellularly and LOXL2 may regulate cell proliferation by enhancing the phosphorylation of PDGFR.

Connective Tissue Growth Factor (CTGF) is a matricellular protein that has been shown to mediate cell adhesion, and as a consequence, it regulates cell proliferation, migration, differentiation and gene transcription. Although previous in vivo and in vitro studies supported the anabolic role of CTGF in skeletogenesis, to date mechanisms of this effect remain unknown. So far, no specific receptor has been identified for CTGF, although previous studies have shown that integrins can serve as functional signaling receptors for CTGF. The CTGF-integrin interaction initiates intracellular signaling cascades that ultimately regulate cell cytoskeleton reorganization, gene transcription and cell function. To study the effect of CTGF on osteoblasts, we first conducted adhesion assays using the MC3T3-E1 osteoblastic cell line. We confirmed that osteoblasts adhere to rCTGF in a concentration-dependent manner and we showed this adhesion has characteristics of integrin mediated adhesions. Next, we used an array of blocking antibodies directed against the individual alpha and beta; integrin subunits that are known to be expressed in osteoblasts. Significant decreases in cell adhesion were observed upon treatment with anti-alpha-v or anti-beta1 blocking antibodies. Subsequent coimmunoprecipitation analyses demonstrated that CTGF interacts with alpha-v and beta1 integrins in osteoblasts. Furthermore, we showed that the specificity of this CTGF-integrin interaction occurs in the C-terminal domain (fourth module) of CTGF. The immunefluorescence staining of cells cultured on substrates of rCTGF, fibronectin (positive control) or BSA (negative control) demonstrated that osteoblast adhesion to rCTGF results in actin cytoskeleton reorganization, focal adhesion formation, enhanced cell spreading and Rac activation. These series of events are necessary for proper cell-matrix interaction and integrins' downstream signaling initiation. Next, through alkaline phosphatase (ALP) staining and activity assays, as well as Alizarin red staining, we demonstrated that osteoblast attachment to CTGF matrix enhances cell maturation, bone nodule formation and matrix mineralization. To investigate whether the effect of CTGF on osteoblast differentiation involves activation of specific signaling molecules, we performed Western blot and chromatin immunoprecipitation (ChIP) assays. Osteoblasts cultured on rCTGF expressed higher levels of both total and phosphorylated forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) compared to the cells cultured on BSA. In addition, these osteoblasts showed an increase in runt-related transcription factor 2 (Runx2) binding to the osteocalcin gene promoter compared to the negative control. These experiments confirmed CTGF's effect on enhancing osteoblast differentiation through regulation of important signaling molecules. In another series of experiments, we used primary osteoblasts isolated from CTGF KO mice, their WT littermates, or WT cells infected to overexpress (OE) CTGF to study the effect of different levels of endogenous CTGF on osteoblast cytoskeleton reorganization and motility. Our assays showed enhanced cell adhesion, spreading and Rac expression in CTGF OE osteoblasts, while in CTGF KO osteoblasts, cell adhesion, spreading and Rac expression were significantly decreased. In contrast, CTGF OE osteoblasts that showed high adhesion and spreading, exhibited diminished cell motility and low levels of RhoA expression, while KO cells migrated quickly and expressed high levels of RhoA. Together, these experiments establish CTGF as an adhesion protein for osteoblasts; they demonstrate that the alpha-v beta1 integrin is a functional signaling receptor for CTGF; they confirm that osteoblast differentiation is enhanced when cultured on CTGF matrix through activation of regulatory signaling molecules; and finally, these experiments establish a role for CTGF in the regulation of small RhoGTPases expression, which in turn implies a significant role for CTGF in cell cytoskeleton reorganization and motility. / Cell Biology

Cutaneous wound healing is a complex process involving the migration of inflammatory cells to the wound site, deposition of extracellular matrix, and the reestablishment of an intact epithelial barrier. Re-epithelialization depends on the proliferation and directional migration of keratinocytes from the wound edges. Initially, keratinocytes migrate over a provisional wound matrix that is rich in fibronectin, and as the wound heals the provisional matrix becomes replaced by one consisting of collagen and proteoglycans. Re-epithelialization is tightly regulated by a variety of peptides such as growth factors, cytokines and proteases, and abnormalities may result in chronic non-healing wounds or hypertrophic scars. CCN2 (Connective Tissue Growth Factor) is a multifunctional protein with effects on cells and their interactions with the connective tissue. CCN2 is expressed in a variety of cell types and regulates numerous cell functions including proliferation, differentiation, adhesion, migration and stimulation of collagen production. While the importance of CCN2 for the fibrotic response has been well studied, its involvement in keratinocyte function has not yet been fully explored. Using an in vivo wound model, the expression of CCN2 was captured at the leading keratinocyte edge during re-epithelialization. In vitro, exogenous addition of CCN2 to human keratinocyte cultures promoted keratinocyte migration. Subsequently, integrin a5b1 was identified as an important mediator of CCN2 enhancement of keratinocyte adhesion to fibronectin. CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1 specific inhibitor PD98059 markedly reduced CCN2-promoted keratinocyte migration. In vitro, CCN2 expression was induced by TGF-β1. Compared with inhibiting the SMAD pathway, blocking MAPK was more effective in reducing TGF-β1-induced CCN2 mRNA and protein expression. In addition, CCN2-induced keratinocyte spreading required FAK. Treatment with CCN2 led to actin disassembly and altered the activity of the Rho proteins and p190RhoGAP in keratinocytes. Furthermore, Cdc42 mediated CCN2-induced cell polarity. In conclusion, using in vivo and in vitro models, CCN2 was shown to regulate keratinocyte function by promoting keratinocyte adhesion, spreading and migration. A complete understanding of CCN2 expression in keratinocytes is crucial in order to develop novel therapies for wound healing.

Connective tissue growth factor (CTGF/CCN2) is axiomatically necessary for proper skeletal development and function. We need not look further than the studies that have been done to date utilizing mice genetically engineered to lack CTGF production. These CTGF null or knockout (KO) mice fail to form a normal murine skeleton and instead yield one littered with bony dysmorphisms, including incompetent craniofacial development, kinked limb bones, and misshapen ribs that are not conducive to proper respiratory function. As a result, the global lack of CTGF is incompatible with postnatal life. A closer look at several sites demonstrated defects in physiologic processes necessary for bone formation - angiogenesis, chondrogenesis, and osteogenesis. Therefore, the dogma in the CCN protein field to date has been that systemic ablation of CTGF production in vivo results in global defects in bone development. We believe this dogma is an oversimplification of the role of CTGF on skeletal development. Our initial impetus leading us to this belief was the gross identification of the specific skeletal sites malformed in CTGF KO mice, in particular the bones of the limbs. While in the lower limb of CTGF KO mice the tibiae and fibulae are misshapen, the adjacent femora and digits are phenotypically normal. The same is true for the upper limb, in which the radii and ulnae are phenotypically abnormal while the humeri and digits are normal. Therefore, we believe that the role of CTGF in skeletogenesis is site-specific such that its loss affects local skeletal patterning and/or mechanobiological cues resulting in the unique phenotype seen in CTGF KO mice. The research of this dissertation constitutes a comprehensive skeletal analysis of CTGF KO mice and in so doing we determined the extent and location of skeletal abnormalities. We found skeletal site-specific changes in growth plate organization, bone microarchitecture and shape and gene expression levels in CTGF KO compared to wild-type (WT) mice. Growth plate malformations included reduced proliferation zone and increased hypertrophic zone lengths. Appendicular skeletal sites demonstrated decreased metaphyseal trabecular bone, while having increased mid-diaphyseal bone and osteogenic expression markers. Axial skeletal analysis showed decreased bone in caudal vertebral bodies, mandibles, and parietal bones in CTGF KO mice, with decreased expression of osteogenic markers. Analysis of skull phenotypes demonstrated global and regional differences in CTGF KO skull shape resulting from allometric (size-based) and non-allometric shape changes. Localized differences in skull morphology included increased skull width and decreased skull length. We further continued the skeletal characterization of CTGF KO bones with an analysis of bone cell ultrastructure and matrix composition. These studies demonstrated that, while CTGF is not necessary for complete morphologic maturation of bone cells, global ablation results in ultrastructural features not commonly seen in WT bones. Our findings include drastically dilated rough endoplasmic reticulum (RER) in osteoblasts of the tibial diaphyseal region, comprising the phenotypic kink in CTGF KO mice and ultrastructural dysmorphologies of CTGF KO osteoclasts including multi-layered, membranous inclusions, decreased vacuolization and ruffled border extents, and disproportionately large clear zones. Lastly, FT-IR analysis demonstrated heterogeneity in CTGF KO bone composition. The results of this dissertation have revealed a more complex role for CTGF in osteogenesis and have identified potential mechanisms and future research directions to fully understand this intricate story. / Cell Biology

Key clinical features of cumulative trauma disorders include pain, muscle weakness, and tissue fibrosis, although the etiology is still under investigation. Therefore, we first sought to characterize the temporal pattern of altered sensorimotor behaviors and inflammatory and fibrogenic processes occurring in forearm muscles and serum of young adult, female rats performing an operant, high repetition high force (HRHF) reaching and grasping task for 6, 12, or 18 weeks. Palmar mechanical sensitivity, cold temperature avoidance and spontaneous behavioral changes increased, while grip strength declined, in 18-week HRHF rats, compared to controls. Flexor digitorum muscles had increased MCP-1 levels after training and increased TNFα in 6-week HRHF rats. Serum had increased IL-1β, IL-10 and IP-10 after training. Yet both muscle and serum inflammation resolved by week 18. In contrast, IFNg increased at week 18 in both muscle and serum. Given the anti-fibrotic role of IFNg, and to identify a mechanism for the continued grip strength losses and behavioral sensitivities, we evaluated the fibrogenic proteins CCN2, collagen type I and TGFß-1, as well as the nociceptive/fibrogenic peptide substance P. Each increased in and around flexor digitorum muscles and extracellular matrix in the mid-forearm, and in nerves of the forepaw at 18 weeks. CCN2 was also increased in serum at week 18. At a time when inflammation had subsided, increases in fibrogenic proteins correlated with sensorimotor declines. Thus, muscle and nerve fibrosis may be critical components of chronic work-related musculoskeletal disorders. CCN2 and substance P may serve as potential targets for therapeutic intervention, and CCN2 as a serum biomarker of fibrosis progression. TGFß-1 and CCN2 are important mediators of tissue fibrosis by their stimulatory effect on extracellular matrix deposition, with CCN2 functions as a downstream mediator of TGFß-1. Substance P (SubP), a nociceptor-related neuropeptide, has also been linked to tissue fibrosis, although little work has been done to understand whether SubP directly causes fibrotic responses in tenocytes. Therefore, we sought to determine if SubP induces fibroblast proliferation and collagen production via CCN2 signaling directly or through the TGFß-1/CCN2 signaling pathway. We hypothesized that SubP may act directly through CCN2, independently from the TGFß-1/CCN2 signaling pathway, to increase fibroblast proliferation and fibrogenic and extracellular matrix protein production in vitro. To examine this question, we assayed cell proliferation and production of CCN2, TGFB1 and collagen type 1 in vitro using primary tendon fibroblasts (tenocytes) isolated from flexor digitorum tendons, and using rat dermal fibroblasts (RDF). We observed that cells isolated from flexor digitorum tendons that express proteins characteristic of tenocytes (vimentin and tenomodulin) underwent increased proliferation in a dose dependent manner after TGFß-1 treatment, but not SubP treatment, as did RDF cells. TGFß-1 treatment increased CCN2 production in both tenocytes and RDF cells, while SubP induced CCN2 production only in rat tenocytes. Expectedly, TGFß-1 treatment increased collagen expression in each cell type, as did SubP treatment alone using In-cell Western analysis. Interestingly, preliminary data that needs to be repeated showed that SubP treatment of each cell type enhanced TGFß-1 expression, assayed using In-cell Western and traditional western blot analyses. Our findings suggest that both SubP and TGFß-1 have distinct fibrogenic actions on tenocytes and dermal fibroblast and that both may be involved in tendinosis observed in animal models and patients with fibrosis. Inflammatory pain, muscle weakness, and tissue fibrosis are key clinical features of work-related musculoskeletal disorders. So, lastly, we evaluated the effects of therapeutic interventions on behavioral and cytokine changes in muscle, tendon and serum of HRHF rats that performed the reaching and grasping task for 11 weeks. We compared sensorimotor behavioral changes, and flexor digitorum tissue inflammation and fibrosis in rats receiving anti-TNFα therapy prophylactically during the initial training, or anti-TNFα therapy with or without rest as secondary interventions during the HRHF work task. Untreated or saline only treated animals at the end of the initial training period had decreased grip strength, increased mechanical sensitivity, and increased serum and tissue inflammatory cytokines (TNFα, IL-1ß, IL-6 and VEGF), changes prevented by prophylactic anti-TNFα treatment. Regarding the secondary interventions, four weeks of anti-TNFα therapy with or without rest, provided in HRHF task weeks 4-7, was more effective than rest alone for restoring grip strength; no treatments rescued forepaw mechanical sensitivity. Effectiveness of the 4-week anti-TNFα therapy extended to week 11, despite no further drug treatment after week 7, for maintenance of grip strength. Tissue cytokine analysis in week 11 showed that HRHF rats treated with saline had increased IL-18 in serum, muscle and tendon, and trends for increased muscle CCN2. Each treatment, particularly anti-TNF with or without rest, decreased serum and tendon IL-18 and IL-1alpha. Rats receiving combined rest and anti-TNFα therapy also had increased serum IL-10. Thus, similar short-term anti-TNFα therapy may be a potential intervention in WMSDs. These results demonstrate that both Substance P and CCN2 play important roles in the development of fibrosis in muscle and tendon in WMSDs based on our model of repetition reaching and grasping. Using in vitro methods, it was demonstrated that substance P is capable of inducing CCN2 in isolated tenocytes and rat dermal fibroblasts, independent of TGFß-1 signaling, a novel discovery that make suggest new treatments for fibrotic disorders. Finally, anti-TNFalpha treatment successfully prevented behavioral declines and increases in IL-18 in serum and tissues in our rat model when provided during the course of HRHF task performance. Key clinical features of cumulative trauma disorders include pain, muscle weakness, and tissue fibrosis, although the etiology is still under investigation. Therefore, we first sought to characterize the temporal pattern of altered sensorimotor behaviors and inflammatory and fibrogenic processes occurring in forearm muscles and serum of young adult, female rats performing an operant, high repetition high force (HRHF) reaching and grasping task for 6, 12, or 18 weeks. Palmar mechanical sensitivity, cold temperature avoidance and spontaneous behavioral changes increased, while grip strength declined, in 18-week HRHF rats, compared to controls. Flexor digitorum muscles had increased MCP-1 levels after training and increased TNFα in 6-week HRHF rats. Serum had increased IL-1β, IL-10 and IP-10 after training. Yet both muscle and serum inflammation resolved by week 18. In contrast, IFNg increased at week 18 in both muscle and serum. Given the anti-fibrotic role of IFNg, and to identify a mechanism for the continued grip strength losses and behavioral sensitivities, we evaluated the fibrogenic proteins CCN2, collagen type I and TGFß-1, as well as the nociceptive/fibrogenic peptide substance P. Each increased in and around flexor digitorum muscles and extracellular matrix in the mid-forearm, and in nerves of the forepaw at 18 weeks. CCN2 was also increased in serum at week 18. At a time when inflammation had subsided, increases in fibrogenic proteins correlated with sensorimotor declines. Thus, muscle and nerve fibrosis may be critical components of chronic work-related musculoskeletal disorders. CCN2 and substance P may serve as potential targets for therapeutic intervention, and CCN2 as a serum biomarker of fibrosis progression. TGFß-1 and CCN2 are important mediators of tissue fibrosis by their stimulatory effect on extracellular matrix deposition, with CCN2 functions as a downstream mediator of TGFß-1. Substance P (SubP), a nociceptor-related neuropeptide, has also been linked to tissue fibrosis, although little work has been done to understand whether SubP directly causes fibrotic responses in tenocytes. Therefore, we sought to determine if SubP induces fibroblast proliferation and collagen production via CCN2 signaling directly or through the TGFß-1/CCN2 signaling pathway. We hypothesized that SubP may act directly through CCN2, independently from the TGFß-1/CCN2 signaling pathway, to increase fibroblast proliferation and fibrogenic and extracellular matrix protein production in vitro. To examine this question, we assayed cell proliferation and production of CCN2, TGFB1 and collagen type 1 in vitro using primary tendon fibroblasts (tenocytes) isolated from flexor digitorum tendons, and using rat dermal fibroblasts (RDF). We observed that cells isolated from flexor digitorum tendons that express proteins characteristic of tenocytes (vimentin and tenomodulin) underwent increased proliferation in a dose dependent manner after TGFß-1 treatment, but not SubP treatment, as did RDF cells. TGFß-1 treatment increased CCN2 production in both tenocytes and RDF cells, while SubP induced CCN2 production only in rat tenocytes. Expectedly, TGFß-1 treatment increased collagen expression in each cell type, as did SubP treatment alone using In-cell Western analysis. Interestingly, preliminary data that needs to be repeated showed that SubP treatment of each cell type enhanced TGFß-1 expression, assayed using In-cell Western and traditional western blot analyses. Our findings suggest that both SubP and TGFß-1 have distinct fibrogenic actions on tenocytes and dermal fibroblast and that both may be involved in tendinosis observed in animal models and patients with fibrosis. Inflammatory pain, muscle weakness, and tissue fibrosis are key clinical features of work-related musculoskeletal disorders. So, lastly, we evaluated the effects of therapeutic interventions on behavioral and cytokine changes in muscle, tendon and serum of HRHF rats that performed the reaching and grasping task for 11 weeks. We compared sensorimotor behavioral changes, and flexor digitorum tissue inflammation and fibrosis in rats receiving anti-TNFα therapy prophylactically during the initial training, or anti-TNFα therapy with or without rest as secondary interventions during the HRHF work task. Untreated or saline only treated animals at the end of the initial training period had decreased grip strength, increased mechanical sensitivity, and increased serum and tissue inflammatory cytokines (TNFα, IL-1ß, IL-6 and VEGF), changes prevented by prophylactic anti-TNFα treatment. Regarding the secondary interventions, four weeks of anti-TNFα therapy with or without rest, provided in HRHF task weeks 4-7, was more effective than rest alone for restoring grip strength; no treatments rescued forepaw mechanical sensitivity. Effectiveness of the 4-week anti-TNFα therapy extended to week 11, despite no further drug treatment after week 7, for maintenance of grip strength. Tissue cytokine analysis in week 11 showed that HRHF rats treated with saline had increased IL-18 in serum, muscle and tendon, and trends for increased muscle CCN2. Each treatment, particularly anti-TNF with or without rest, decreased serum and tendon IL-18 and IL-1alpha. Rats receiving combined rest and anti-TNFα therapy also had increased serum IL-10. Thus, similar short-term anti-TNFα therapy may be a potential intervention in WMSDs. These results demonstrate that both Substance P and CCN2 play important roles in the development of fibrosis in muscle and tendon in WMSDs based on our model of repetition reaching and grasping. Using in vitro methods, it was demonstrated that substance P is capable of inducing CCN2 in isolated tenocytes and rat dermal fibroblasts, independent of TGFß-1 signaling, a novel discovery that make suggest new treatments for fibrotic disorders. Finally, anti-TNFalpha treatment successfully prevented behavioral declines and increases in IL-18 in serum and tissues in our rat model when provided during the course of HRHF task performance. / Cell Biology