Ultraviolet A-Induced Cathepsin K Expression Is Mediated via MAPK/AP-1 Pathway in Human Dermal Fibroblasts

Background

Cathepsin K (CatK), a cysteine protease with the potent elastolytic activity, plays a predominant role in intracellular elastin degradation in human dermal fibroblasts (HDFs), and contributes to solar elastosis. In previous studies, CatK expression was downregulated in photoaged skin and fibroblasts, but upregulated in acute UVA-irradiated skin and fibroblasts. The underlying mechanisms regulating UVA-induced CatK expression remain elusive.

Objective

This study investigates mechanisms involved in the regulation of UVA-induced CatK expression in HDFs.

Methods

Primary HDFs were exposed to UVA. Cell proliferation was analyzed using a colorimetric assay of relative cell number. Quantitative real-time RT-PCR and Western blot were performed to detect CatK expression in HDFs on three consecutive days after 10 J/cm² UVA irradiation, or cells treated with increasing UVA doses. UVA-activated MAPK/AP-1 pathway was examined by Western blot. Effects of inhibition of MAPK pathway and knockdown of Jun and Fos on UVA-induced CatK expression were also measured by real-time RT-PCR and Western blot.

Results

UVA significantly increased CatK mRNA and protein expression in a dose-dependent manner. UVA-induced CatK expression occurred along with UVA-activated phosphorylation of JNK, p38 and Jun, UVA-increased expression of Fos. Inactivation of JNK and p38MAPK pathways both remarkably decreased UVA-induced CatK expression, which was suppressed more by inhibition of JNK pathway. Furthermore, knockdown of Jun and Fos significantly attenuated basal and UVA-induced CatK expression.

Conclusion

UVA is capable of increasing CatK expression in HDFs, most likely by activation of MAPK pathway and of AP-1, which has been shown to be the case for matrix metalloproteinases. As current strategies for selecting anti-photoaging agents focus on their ability to decrease MMPs'' expression through inhibiting UV- activated MAPK pathway, future strategies should also consider their effect on CatK expression.     

Toll-like receptor 3 upregulation by type I interferon in healthy and scleroderma dermal fibroblasts

Introduction  

Increased levels of genes in the type I interferon (IFN) pathway have been observed in patients with systemic sclerosis (SSc), or scleroderma. How type I IFN regulates the dermal fibroblast and its participation in the development of dermal fibrosis is not known. We hypothesized that one mechanism by which type I IFN may contribute to dermal fibrosis is through upregulation of specific Toll-like receptors (TLRs) on dermal fibroblasts. Therefore, we investigated the regulation of TLR expression on dermal fibroblasts by IFN.     

Contributions of Ocular Surface Components to Matrix-Metalloproteinases (MMP)-2 and MMP-9 in Feline Tears following Corneal Epithelial Wounding

Purpose

This study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding.

Methods

Laboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs.

Results

The proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (p<0.05). Tears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining.

Conclusions

Following wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following corneal wounding are the lacrimal gland and CALT. Other sources included stromal keratocytes and conjunctiva with goblet cells.     

Effect of experimental treatment on GAPDH mRNA expression as a housekeeping gene in human diploid fibroblasts

Background  

Several genes have been used as housekeeping genes and choosing an appropriate reference gene is important for accurate quantitative RNA expression in real time RT-PCR technique. The expression levels of reference genes should remain constant between the cells of different tissues and under different experimental conditions. The purpose of this study was to determine the effect of different experimental treatments on the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA so that the reliability of GAPDH as reference gene for quantitative real time RT-PCR in human diploid fibroblasts (HDFs) can be validated. HDFs in 4 different treatment groups viz; young (passage 4), senescent (passage 30), H₂O₂-induced oxidative stress and γ-tocotrienol (GTT)-treated groups were harvested for total RNA extraction. Total RNA concentration and purity were determined prior to GAPDH mRNA quantification. Standard curve of GAPDH expression in serial diluted total RNA, melting curve analysis and agarose gel electrophoresis were used to determine the reliability of GAPDH as reference gene.     

Impaired wound healing in mice deficient in a matricellular protein SPARC (osteonectin, BM-40)

Background  

SPARC is a matricellular protein involved in cell-matrix interactions. From expression patterns at the wound site and in vitro studies, SPARC has been implicated in the control of wound healing. Here we examined the function of SPARC in cutaneous wound healing using SPARC-null mice and dermal fibroblasts derived from them.     

Suppression of MMP-9 by doxycycline in brain arteriovenous malformations

Background  

The primary aim of this study is to demonstrate the feasibility of utilizing doxycycline to suppress matrix metalloproteinase-9 (MMP-9) in brain arteriovenous malformations (AVMs).     

Thrombin induces macrophage migration inhibitory factor release and upregulation in urothelium: a possible contribution to bladder inflammation

Purpose

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine expressed by urothelial cells that mediates bladder inflammation. We investigated the effect of stimulation with thrombin, a Protease Activated Receptor-1 (PAR1) agonist, on MIF release and MIF mRNA upregulation in urothelial cells.

Materials and Methods

MIF and PAR1 expression was examined in normal human immortalized urothelial cells (UROtsa) using real-time RT-PCR, Western blotting and dual immunostaining. MIF and PAR1 immunostaining was also examined in rat urothelium. The effect of thrombin stimulation (100 nM) on urothelial MIF release was examined in UROtsa cells (in vitro) and in rats (in vivo). UROtsa cells were stimulated with thrombin, culture media were collected at different time points and MIF amounts were determined by ELISA. Pentobarbital anesthetized rats received intravesical saline (control), thrombin, or thrombin +2% lidocaine (to block nerve activity) for 1 hr, intraluminal fluid was collected and MIF amounts determined by ELISA. Bladder or UROtsa MIF mRNA was measured using real time RT-PCR.

Results

UROtsa cells constitutively express MIF and PAR1 and immunostaining for both was observed in these cells and in the basal and intermediate layers of rat urothelium. Thrombin stimulation of urothelial cells resulted in a concentration- and time-dependent increase in MIF release both in vitro (UROtsa; 2.8-fold increase at 1 hr) and in vivo (rat; 4.5-fold) while heat-inactivated thrombin had no effect. In rats, thrombin-induced MIF release was reduced but not abolished by intravesical lidocaine treatment. Thrombin also upregulated MIF mRNA in UROtsa cells (3.3-fold increase) and in the rat bladder (2-fold increase) where the effect was reduced (1.4-fold) by lidocaine treatment.

Conclusions

Urothelial cells express both MIF and PAR1. Activation of urothelial PAR1 receptors, either by locally generated thrombin or proteases present in the urine, may mediate bladder inflammation by inducing urothelial MIF release and upregulating urothelial MIF expression.     

Arousal of cancer-associated stroma: overexpression of palladin activates fibroblasts to promote tumor invasion

Background

Cancer-associated fibroblasts, comprised of activated fibroblasts or myofibroblasts, are found in the stroma surrounding solid tumors. These myofibroblasts promote invasion and metastasis of cancer cells. Mechanisms regulating the activation of the fibroblasts and the initiation of invasive tumorigenesis are of great interest. Upregulation of the cytoskeletal protein, palladin, has been detected in the stromal myofibroblasts surrounding many solid cancers and in expression screens for genes involved in invasion. Using a pancreatic cancer model, we investigated the functional consequence of overexpression of exogenous palladin in normal fibroblasts in vitro and its effect on the early stages of tumor invasion.

Principal Findings

Palladin expression in stromal fibroblasts occurs very early in tumorigenesis. In vivo, concordant expression of palladin and the myofibroblast marker, alpha smooth muscle actin (α-SMA), occurs early at the dysplastic stages in peri-tumoral stroma and progressively increases in pancreatic tumorigenesis. In vitro introduction of exogenous 90 kD palladin into normal human dermal fibroblasts (HDFs) induces activation of stromal fibroblasts into myofibroblasts as marked by induction of α-SMA and vimentin, and through the physical change of cell morphology. Moreover, palladin expression in the fibroblasts enhances cellular migration, invasion through the extracellular matrix, and creation of tunnels through which cancer cells can follow. The fibroblast invasion and creation of tunnels results from the development of invadopodia-like cellular protrusions which express invadopodia proteins and proteolytic enzymes. Palladin expression in fibroblasts is triggered by the co-culture of normal fibroblasts with k-ras-expressing epithelial cells.

Conclusions

Overall, palladin expression can impart myofibroblast properties, in turn promoting the invasive potential of these peri-tumoral cells with invadopodia-driven degradation of extracellular matrix. Palladin expression in fibroblasts can be triggered by k-ras expression in adjacent epithelial cells. This data supports a model whereby palladin-activated fibroblasts facilitate stromal-dependent metastasis and outgrowth of tumorigenic epithelium.     

B lymphocytes and B-cell activating factor promote collagen and profibrotic markers expression by dermal fibroblasts in systemic sclerosis

Introduction

B lymphocytes might play a pathogenic role in dermal fibrosis in systemic sclerosis (SSc). B-cell activating factor (BAFF), a key cytokine for B-cell activation, is increased in the serum and the skin of patients with SSc. However, the ability of B cells directly to stimulate dermal fibroblasts and the role of BAFF are not fully understood. We therefore investigated the involvement of B cells and BAFF in the expression of collagen and profibrotic markers by dermal fibroblasts.

Methods

Cocultures of blood B cells from healthy blood donors and normal or SSc dermal fibroblasts stimulated with anti-IgM and BAFF were performed. Alpha-SMA, TIMP1, MMP9, COL1A1, COL1A2, and COL3A1 mRNA expression were determined by quantitative RT-PCR. Soluble collagen, BAFF, IL-6, IL-1β, TGF-β1, and CCL2 protein secretion were assessed.

Results

Coculture of blood B cells and dermal fibroblasts isolated from SSc patients induced IL-6, TGF-β1, CCL2, and collagen secretion, as well as Alpha-SMA, TIMP1, and MMP9 expression in dermal fibroblasts. Transwell assays demonstrated that this induction was dependent on cell-cell contact. Addition of anti-IgM and BAFF to the coculture increased IL-6, CCL2, TGF-β1, and collagen secretion. B cell- and BAFF-induced collagen secretion was highly reduced by anti-TGF-β1 antibodies.

Conclusions

Our results showed for the first time a direct role of B cells on the production of collagen by dermal fibroblasts, which is further enhanced by BAFF. Thus, these results demonstrate a new pathogenic role of B cells and BAFF in fibrosis and systemic sclerosis.     

Electronegative LDL induces MMP-9 and TIMP-1 release in monocytes through CD14 activation: Inhibitory effect of glycosaminoglycan sulodexide

Objective

Electronegative LDL (LDL(?)) is involved in atherosclerosis through the activation of the TLR4/CD14 inflammatory pathway in monocytes. Matrix metalloproteinases (MMP) and their inhibitors (tissue inhibitors of metalloproteinase [TIMP]) are also crucially involved in atherosclerosis, but their modulation by LDL(?) has never been investigated. The aim of this study was to examine the ability of LDL(?) to release MMPs and TIMPs in human monocytes and to determine whether sulodexide (SDX), a glycosaminoglycan-based drug, was able to affect their secretion.

Growth factors‐based platelet lysate rejuvenates skin against ageing through NF‐κB signalling pathway: In vitro and in vivo mechanistic and clinical studies

IntroductionPlatelets benefit tissue regeneration by secreting growth factors, and platelet products, for example, platelet lysate (PL), have been clinically applied for tissue rejuvenation. To determine the anti‐ageing efficacy and mechanism of human PL (hPL) on skin, this study conducted clinical retrospective analysis, nude mice‐based in vivo study and human dermal fibroblasts (HDFs)‐based in vitro study.MethodsFlow cytometry was employed for quality control of hPL, and ELISA was used for quantification of growth factors (EGF, IGF‐1, PDGF and TGF‐β) in hPL. After d‐galactose modelling, skin texture grading, histopathological observation, immunofluorescence analysis and oxidative stress assays were conducted on nude mice, while SA‐β‐gal staining, CCK‐8 and wound healing assays were conducted on HDFs. qPCR and western blot were conducted to clarify hPL''s mechanism.ResultsThe clinical retrospective data showed that hPL obviously rejuvenated human skin appearances without adverse events. The animal data showed that hPL exerted rejuvenative effects on skin, and the cellular data showed that hPL significantly promoted the proliferation and migration of HDFs and suppressed senescence‐associated secretory protein secretion and senescence state of senescent HDFs by suppressing NF‐κB pathway. The NF‐κB‐dependent mechanism was verified positively by using P65 siRNA and negatively by using prostratin. Furthermore, EGF, IGF‐1, PDGF and TGF‐β were found as the main ingredients in hPL, which contributed to the efficacy and mechanism of hPL.ConclusionThis study provided novel knowledge of hPL, making it ideal for skin rejuvenation.

Human platelet lysate (hPL) exerted rejuvenative and regenerative efficacy on skin ageing of nude mice and senescent HDFs through NF‐κB signalling pathway. A retrospective study further verified hPL''s efficacy and safety in clinic.     

Involvement of CD147 in overexpression of MMP-2 and MMP-9 and enhancement of invasive potential of PMA-differentiated THP-1

Background  

During infection and inflammation, circulating blood monocytes migrate from the intravascular compartments to the extravascular compartments, where they mature into tissue macrophages. The maturation process prepares the cells to actively participate in the inflammatory and immune responses, and many factors have been reported to be involved in the process. We found in our study that CD147 played a very important role in this process.     

Promotive effects of human induced pluripotent stem cell-conditioned medium on the proliferation and migration of dermal fibroblasts

Fibroblasts are a major cell type in the dermis. When skin is wounded in various ways such as by abrasions, cuts or diabetic ulcer, proliferation and migration of dermal fibroblasts is necessary for cutaneous wound healing. Numerous studies have shown that adult stem cells secrete paracrine factors and these are able to promote wound healing by activating migration and proliferation of effector cells such as dermal fibroblasts. However, the paracrine factors secreted from pluripotent stem cells and the effect of these on dermal fibroblast proliferation and migration have been poorly characterized. In this study we cultured human induced pluripotent stem cells without any animal-derived components including feeder cells, and investigated the effect of stem cell-conditioned medium (iPSC-CM) on dermal fibroblast proliferation and migration. Results showed that the proliferation of mouse embryonic fibroblasts (STO cells) and human dermal fibroblasts (HDFs) were significantly stimulated by iPSC-CM. We determined that the optimal concentration of iPSC-CM in promoting the proliferation of HDFs was a 75% dilution. Scratch wound assay and transwell migration assay also demonstrated the stimulatory effect of iPSC-CM on the migration of HDFs. iPSC-CM is believed to have advantages because of the unique capabilities of iPSCs, which include infinite self-renewal, pluripotency and variety of donor cells. Thus, iPSC-CM is anticipated to be a valuable source of paracrine factors which can potentially be used for wound healing applications.     

Glucocorticosteroids differentially regulate MMP-9 and neutrophil elastase in COPD

Background

Chronic Obstructive Pulmonary Disease (COPD) is currently the fifth leading cause of death worldwide. Neutrophilic inflammation is prominent, worsened during infective exacerbations and is refractory to glucocorticosteroids (GCs). Deregulated neutrophilic inflammation can cause excessive matrix degradation through proteinase release. Gelatinase and azurophilic granules within neutrophils are a major source of matrix metalloproteinase (MMP)-9 and neutrophil elastase (NE), respectively, which are elevated in COPD.

Methods

Secreted MMP-9 and NE activity in BALF were stratified according to GOLD severity stages. The regulation of secreted NE and MMP-9 in isolated blood neutrophils was investigated using a pharmacological approach. In vivo release of MMP-9 and NE in mice exposed to cigarette smoke (CS) and/or the TLR agonist lipopolysaccharide (LPS) in the presence of dexamethasone (Dex) was investigated.

Results

Neutrophil activation as assessed by NE release was increased in severe COPD (36-fold, GOLD II vs. IV). MMP-9 levels (8-fold) and activity (21-fold) were also elevated in severe COPD, and this activity was strongly associated with BALF neutrophils (r = 0.92, p<0.001), but not macrophages (r = 0.48, p = 0.13). In vitro, release of NE and MMP-9 from fMLP stimulated blood neutrophils was insensitive to Dex and attenuated by the PI3K inhibitor, wortmannin. In vivo, GC resistant neutrophil activation (NE release) was only seen in mice exposed to CS and LPS. In addition, GC refractory MMP-9 expression was only associated with neutrophil activation.

Conclusions

As neutrophils become activated with increasing COPD severity, they become an important source of NE and MMP-9 activity, which secrete proteinases independently of TIMPs. Furthermore, as NE and MMP-9 release was resistant to GC, targeting of the PI3K pathway may offer an alternative pathway to combating this proteinase imbalance in severe COPD.