CCN2 is transiently expressed by keratinocytes during re-epithelialization and regulates keratinocyte migration in vitro by the ras-MEK-ERK signaling pathway

Background

CCN2 (previously known as connective tissue growth factor) is a multifunctional matricellular protein that has numerous effects on cell life and cell interactions with the connective tissue. Although the importance of CCN2 for the fibrotic process in wound healing has been well studied, the involvement of CCN2 in keratinocyte function has not yet been explored. Therefore, the aim of the present study was to investigate the role of CCN2 in the epidermis during wound healing.

Materials and methods

Immunohistochemistry was done on sections from full-thickness porcine wounds. The effect of CCN2 on the migration of cultured human keratinocytes exposed to scratch wounds, the effect on phosphorylation of extracellular signal-related kinases (ERK), and the effect of adding inhibitors to the ERK/mitogen-activated protein kinase pathway to human keratinocytes were studied.

Results

The CCN2 protein was transiently expressed in vivo at the leading keratinocyte edge during re-epithelialization of full-thickness porcine wounds. In vitro, exogenous addition of CCN2 to human keratinocyte cultures regulated keratinocyte migration and resulted in phosphorylation of ERK. The addition of inhibitors of ERK/mitogen-activated protein kinase counteracted the effect of CCN2 on migration.

Conclusions

CCN2 was transiently expressed at the leading keratinocyte edge in vivo. The biologic importance of this was supported in vitro, because CCN2 regulated human keratinocyte migration through activation of the Ras-mitogen-activated protein kinase kinase-ERK signal transduction pathway.     

Cdc42 and p190RhoGAP activation by CCN2 regulates cell spreading and polarity and induces actin disassembly in migrating keratinocytes

Cell migration requires spatiotemporal integration of signals that regulate cytoskeletal dynamics. In response to a migration‐promoting agent, cells begin to polarise and extend protrusions in the direction of migration. These cytoskeletal rearrangements are orchestrated by a variety of proteins, including focal adhesion kinase (FAK) and the Rho family of GTPases. CCN2, also known as connective tissue growth factor, has emerged as a regulator of cell migration but the mechanism by which CCN2 regulates keratinocyte function is not well understood. In this article, we sought to elucidate the basic mechanism of CCN2‐induced cell migration in human keratinocytes. Immunohistochemical staining was used to demonstrate that treatment with CCN2 induces a migratory phenotype through actin disassembly, spreading of lamellipodia and re‐orientation of the Golgi. In vitro assays were used to show that CCN2‐induced cell migration is dependent on FAK, RhoA and Cdc42, but independent of Rac1. CCN2‐treated keratinocytes displayed increased Cdc42 activity and decreased RhoA activity up to 12 hours post‐treatment, with upregulation of p190RhoGAP. An improved understanding of how CCN2 regulates cell migration may establish the foundation for future therapeutics in fibrotic and neoplastic diseases.     

Inhibition of cyclooxygenase‐1 and ‐2 activity in keratinocytes inhibits PGE2 formation and impairs vascular endothelial growth factor release and neovascularisation in skin wounds

Inhibition of cyclooxygenase (Cox) enzymatic activity by non‐steroidal anti‐inflammatory drugs (NSAIDs) provides the molecular basis of analgesia following wounding or surgery. This study investigated the role of Cox activity in the regulation of vascular endothelial growth factor (VEGF) expression in keratinocytes and the formation of new blood vessels in acute wounds in mice. To this end, human HaCaT keratinocytes were stimulated with epidermal growth factor (EGF). EGF increased Cox‐1 mRNA in the presence of the constitutively expressed Cox‐1 protein in keratinocytes. EGF coinduced Cox‐2 and VEGF₁₆₅ mRNA and protein expression and an accumulation of prostaglandin E₂ (PGE₂) in cell culture supernatants. Inhibition of Cox isozyme activity by Cox‐1 and ‐2 siRNA or ibuprofen reduced PGE₂ and VEGF₁₆₅ release from keratinocytes. In a mouse model of excisional wound healing, Cox‐2 and VEGF₁₆₅ expression were colocalized in the granulation tissue of acute wounds. Oral treatment of mice with the Cox‐1 and ‐2 inhibitor diclofenac was associated with reduced levels of VEGF₁₆₅ protein and an impaired blood vessel formation in acute wound tissue. In summary, our data suggest that a reduction of PGE₂‐triggered VEGF₁₆₅ protein expression in wound keratinocytes is likely to contribute to the observed impairment of wound neovascularisation upon Cox inhibition.     

Modulation of wound contracture α‐smooth muscle actin and multispecific vitronectin receptor integrin αvβ3 in the rabbit's experimental model

The myofibroblast, a major component of granulation tissue, is a key cell during wound healing, tissue repair and connective tissue remodelling. Persistence of myofibroblasts within a fibrotic lesion leads to excessive scarring impairing function and aesthetics. Various wound‐healing cytokines can be modulated by topical application of active agents to promote optimal wound healing and improve scar quality. Thus, the myofibroblast may represent an important target for wound‐healing modulation to improve the evolution of conditions such as hypertrophic scars. The purpose of this work is to study the modulation of myofibroblasts and integrin αvβ3 in a full thickness wound performed on rabbits treated with different topical agents using: (1) saline, (2) Tegaderm occlusive dressing (3) silver sulfadiazine and (4) moist exposed burn ointment (MEBO). The reepithelialisation was 4 days faster in the MEBO group compared with the other therapies with less oedema formation, delayed contraction, less inflammatory cells and the lowest transepidermal water loss (TEWL) resulting in a soft scar. Although α‐smooth muscle actin (α‐SMA) was the highest around day 12 in the MEBO group, wound contraction and myofibroblast's activity were the least for the same period probably because of a downregulation of the integrin αvβ3. It seems that the effect of MEBO could be more pronounced on force transmission rather then on force generation. Greater insight into the pathology of scars may translate into non surgical treatments in the future and further work in myofibroblast biology will eventually result in efficient pharmacological tools, improving the evolution of healing and scar formation.     

Compressive Force‐Produced CCN2 Induces Osteocyte Apoptosis Through ERK1/2 Pathway

Osteocytes produce various factors that mediate the onset of bone formation and resorption and play roles in maintaining bone homeostasis and remodeling in response to mechanical stimuli. One such factor, CCN2, is thought to play a significant role in osteocyte responses to mechanical stimuli, but its function in osteocytes is not well understood. Here, we showed that CCN2 induces apoptosis in osteocytes under compressive force loading. Compressive force increased CCN2 gene expression and production, and induced apoptosis in osteocytes. Application of exogenous CCN2 protein induced apoptosis, and a neutralizing CCN2 antibody blocked loading‐induced apoptosis. We further examined how CCN2 induces loaded osteocyte apoptosis. In loaded osteocytes, extracellular signal‐regulated kinase 1/2 (ERK1/2) was activated, and an ERK1/2 inhibitor blocked loading‐induced apoptosis. Furthermore, application of exogenous CCN2 protein caused ERK1/2 activation, and the neutralizing CCN2 antibody inhibited loading‐induced ERK1/2 activation. Therefore, this study demonstrated for the first time to our knowledge that enhanced production of CCN2 in osteocytes under compressive force loading induces apoptosis through activation of ERK1/2 pathway. © 2014 American Society for Bone and Mineral Research.     

CCN5 Reduces Ligamentum Flavum Hypertrophy by Modulating the TGF‐β Pathway

Ligamentum flavum hypertrophy (LFH) is the most important component of lumbar spinal canal stenosis. Although the pathophysiology of LFH has been extensively studied, no method has been proposed to prevent or treat it. Since the transforming growth factor‐β (TGF‐β) pathway is known to be critical in LFH pathology, we investigated whether LFH could be prevented by blocking or modulating the TGF‐β mechanism. Human LF cells were used for the experiments. First, we created TGF‐β receptor 1 (TGFBR1) knock out (KO) cells with CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 biotechnology and treated them with TGF‐β1 to determine the effects of blocking the TGF‐β pathway. Subsequently, we studied the effect of CCN5, which has recently been proposed to modulate the TGF‐β pathway. To assess the predisposition toward fibrosis, α‐smooth muscle actin (αSMA), fibronectin, collagen‐1, collagen‐3, and CCN2 were evaluated with quantitative real‐time polymerase chain reaction, western blotting, and immunocytochemistry. The TGFBR1 KO LF cells were successfully constructed with high KO efficiency. In wild‐type (WT) cells, treatment with TGF‐β1 resulted in the overexpression of the messenger RNA (mRNA) of fibrosis‐related factors. However, in KO cells, the responses to TGF‐β1 stimulation were significantly lower. In addition, CCN5 and TGF‐β1 co‐treatment caused a notable reduction in mRNA expression levels compared with TGF‐β1 stimulation only. The αSMA protein expression increased with TGF‐β1 but decreased with CCN5 treatment. TGF‐β1 induced LF cell transdifferentiation from fibroblasts to myofibroblasts. However, this cell transition dramatically decreased in the presence of CCN5. In conclusion, CCN5 could prevent LFH by modulating the TGF‐β pathway. © 2019 The Authors. Journal of Orthopaedic Research^® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:2634–2644, 2019     

Interleukin‐1β stimulates stromal‐derived factor‐1α expression in human subacromial bursa

Chemokines produced by synoviocytes of the subacromial bursa are up‐regulated in subacromial bursitis and rotator cuff disease. We hypothesized that SDF‐1α production in bursal synoviocytes may be induced by local cytokines such as interleukin IL‐1β and IL‐6. Subacromial bursa specimens were obtained from patients undergoing shoulder surgery. Bursal specimens were stained with anti‐human antibodies to IL‐1, IL‐6, and SDF‐1α by immunohistochemistry and compared to normal and rheumatoid controls. Bursal cells were also isolated from specimens and cultured. Early passaged cells were then treated with cytokines (IL‐1β and IL‐6) and SDF‐1α expression was measured by ELISA and RT‐PCR. SDF‐1α, IL‐1β, and IL‐6 were expressed at high levels in bursitis specimens from human subacromial bursa compared to normal controls. In cultured bursal synoviocytes, there was a dose‐dependent increase in SDF‐1α production in the supernatants of cells treated with IL‐1β. SDF‐1α mRNA expression was also increased in bursal cells treated with IL‐1β. IL‐6 caused a minimal but not statistically significant increase in SDF‐1α expression. SDF‐1α, IL‐1β, and IL‐6 are expressed in the inflamed human subacromial bursal tissues in patients with subacromial bursitis. In cultured bursal synoviocytes, SDF‐1α gene expression and protein production are stimulated by IL‐1β. IL‐1β produced by bursal syvoviocytes and inflammatory cells in the human subacromial bursa is an important signal in the inflammatory response that occurs in subacromial bursitis and rotator cuff disease. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1695–1699, 2011     

Ⅱ度烧伤病人皮肤角朊细胞ICAM-1的表达

目的探讨Ⅱ度烧伤创面愈合过程中角朊细胞间粘附分子-1(ICAM-1)的表达及其意义。方法应用免疫组织化学染色技术,对烧伤后不同时间Ⅱ度烧伤皮肤组织角朊细胞 ICAM-1的表达进行动态观察。8例健康人皮肤,6例烧伤病人正常皮肤及8例Ⅱ度烧伤愈合后增生性瘢痕组织为对照组。结果健康人正常皮肤基底层细胞 ICAM-1可低表达,烧伤病人正常皮肤基底层细胞ICAM-1可表达增强。Ⅱ度创面基底层细胞伤后1周内 ICAM-1表达轻度增高,2周内明显增强,2～4周稳定高表达,ICAM-1呈极性分布,新生上皮多层角朊细胞 ICAM-1表达显著增强,ICAM-1阳性细胞呈长柱状排列。结论Ⅱ度烧伤皮肤角朊细胞 ICAM-1表达增强可能与创基炎性细胞浸润相关,具有诱导角朊细胞增殖、迁移,加速上皮化的作用。     

Transforming growth factor‐β (TGF‐β) expression is increased in the subsynovial connective tissues of patients with idiopathic carpal tunnel syndrome

Non‐inflammatory fibrosis of the subsynovial connective tissue (SSCT) is a hallmark of carpal tunnel syndrome (CTS). The etiology of this finding and its relationship to the development of CTS remain poorly understood. Recent studies have found that transforming growth factor‐β (TGF‐β) plays a central role in fibrosis. The purpose of this study was to investigate the expression of TGF‐β and connective tissue growth factor (CTGF), a downstream mediator of TGF‐β, in the pathogenesis of CTS. We compared SSCT specimens from 26 idiopathic CTS patients with specimens from 10 human cadaver controls with no previous diagnosis of CTS. Immunohistochemistry was performed to determine levels TGF‐β1, CTGF, collagen 1(Col1) and collagen 3 (Col3) expression. TGF‐β1 (p < 0.01), CTGF (p < 0.01), and Col3 (p < 0.01) were increased in SSCT of CTS patients compared with control tissue. In addition, a strong positive correlation was found between TGF‐β1 and CTGF, (R² = 0.80, p < 0.01) and a moderate positive correlation between Col3 and TGF‐β1 (R² = 0.49, p < 0.01). These finding suggest that there is an increased expression of TGF‐β and CTGF, a TGF‐β regulated protein, and that this TGF‐β activation may be responsible for SSCT fibrosis in CTS patients. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:116–122, 2014.     

CCN family 2/connective tissue growth factor (CCN2/CTGF) promotes osteoclastogenesis via induction of and interaction with dendritic cell–specific transmembrane protein (DC‐STAMP)

CCN family 2/connective tissue growth factor (CCN2/CTGF) promotes endochondral ossification. However, the role of CCN2 in the replacement of hypertrophic cartilage with bone is still unclear. The phenotype of Ccn2 null mice, having an expanded hypertrophic zone, indicates that the resorption of the cartilage extracellular matrix is impaired therein. Therefore, we analyzed the role of CCN2 in osteoclastogenesis because cartilage extracellular matrix is resorbed mainly by osteoclasts during endochondral ossification. Expression of the Ccn2 gene was upregulated in mouse macrophage cell line RAW264.7 on day 6 after treatment of glutathione S transferase (GST) fusion mouse receptor activator of NF‐κB ligand (GST‐RANKL), and a combination of recombinant CCN2 (rCCN2) and GST‐RANKL significantly enhanced tartrate‐resistant acid phosphatase (TRACP)–positive multinucleated cell formation compared with GST‐RANKL alone. Therefore, we suspected the involvement of CCN2 in cell‐cell fusion during osteoclastogenesis. To clarify the mechanism, we performed real‐time PCR analysis of gene expression, coimmunoprecipitation analysis, and solid‐phase binding assay of CCN2 and dendritic cell–specific transmembrane protein (DC‐STAMP), which is involved in cell‐cell fusion. The results showed that CCN2 induced and interacted with DC‐STAMP. Furthermore, GST‐RANKL–induced osteoclastogenesis was impaired in fetal liver cells from Ccn2 null mice, and the impaired osteoclast formation was rescued by the addition of exogenous rCCN2 or the forced expression of DC‐STAMP by a retroviral vector. These results suggest that CCN2 expressed during osteoclastogenesis promotes osteoclast formation via induction of and interaction with DC‐STAMP. © 2011 American Society for Bone and Mineral Research.     

Hyaluronic acid modulates gene expression of connective tissue growth factor (CTGF), transforming growth factor‐β1 (TGF‐β1), and vascular endothelial growth factor (VEGF) in human fibroblast‐like synovial cells from advanced‐stage osteoarthritis in vitro

Intraarticular injection of hyaluronan (hyaluronic acid; HA) is the common way to treat osteoarthritis (OA) of knees. This treatment cannot only maintain the viscoelastic properties of knee but also release the OA pain. However, the exact molecular mechanism is unknown. In this study, after human synovial cells were stimulated with HA and Hylan (Synvisc®) for 24 h, real‐time polymerase chain reaction (real‐time PCR) was used to detect the alteration of connective tissue growth factor (CTGF), transforming growth factor‐β1 (TGF‐β1), and vascular endothelial growth factor (VEGF) gene expression, which were specific genes related to pathogenesis of OA knees. Our results illustrated that both HA and Hylan might not cause cytotoxicity or apoptosis of synovial cells in serum deprivation environment. The gene expressions of TGF‐β1 and VEGF were significantly increased at the concentration of 0.1 mg/mL HA and 0.1 mg/mL Hylan, respectively (α < 0.05). The synovial cells with treatment of 0.1 mg/mL Hylan decreased the CTGF gene expression (0.66‐fold) and VEGF (0.78‐fold) compared to 0.1 mg/mL HA (α < 0.05). We suggested that the profile of CTGF, TGF‐β1, and VEGF gene expressions in our study might provide the rational mechanism for the therapeutic effect of hyaluronan on OA knees. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:492–496, 2010     

Fibronectin‐α4β1 Interactions in Hepatic Cold Ischemia and Reperfusion Injury: Regulation of MMP‐9 and MT1‐MMP via the p38 MAPK Pathway

Liver ischemia‐reperfusion injury (IRI) remains a challenging problem in clinical settings. The expression of fibronectin (FN) by endothelial cells is a prominent feature of the hepatic response to injury. Here we investigate the effects of the connecting segment‐1 (CS‐1) peptide therapy, which blocks FN‐α4β1 integrin leukocyte interactions, in a well‐established model of 24‐h cold liver IRI. CS‐1 peptides significantly inhibited leukocyte recruitment and local release of proinflammatory mediators (COX‐2, iNOS and TNF‐α), ameliorating liver IRI and improving recipient survival rate. CS1 therapy inhibited the phosphorylation of p38 MAPK, a kinase linked to inflammatory processes. Moreover, in addition to downregulating the expression of matrix metalloproteinase‐9 (MMP‐9) in hepatic IRI, CS‐1 peptide therapy depressed the expression of membrane type 1‐matrix metalloproteinase (MT1‐MMP/MMP‐14) by macrophages, a membrane‐tethered MMP important for focal matrix proteolysis. Inhibition of p38 MAPK activity, with its pharmacological antagonist SB203580, downregulated MMP‐9 and MT1‐MMP/MMP‐14 expressions by FN‐stimulated macrophages, suggesting that p38 MAPK kinase pathway controls FN‐mediated inductions of MMP‐9 and MT1‐MMP/MMP‐14. Hence, this study provides new insights on the role of FN in liver injury, which can potentially be applied to the development of new pharmacological strategies for the successful protection against hepatic IRI.