In vitro induction of matrix metalloproteinase-2 and matrix metalloproteinase-9 expression in keratinocytes by boron and manganese

Matrix metalloproteinase (MMP)-2 and MMP-9 are involved in keratinocyte migration and granulation tissue remodeling during wound healing. Thermal water cures are sometimes proposed as complementary treatment for accelerating healing of wounds resulting from burns and/or surgery, but their mechanisms of action remain unknown. Some thermal waters are rich in trace elements such as boron and manganese. Interestingly, clinical studies have shown the beneficial effects of trace elements such as boron and manganese for human wound healing. To try to specify the role of trace elements in cutaneous healing, the present study investigated the effects of these trace elements on the production of MMP-2 and MMP-9 by normal human keratinocytes cultured in vitro. Immunohistochemistry and Western blot showed that intracellular MMP-9 expression in keratinocytes was induced when incubated for 6 h with boron at 10 micro g/ml or manganese at 0.2 micro g/ml. Moreover, gelatin zymography on keratinocyte supernatants showed an increase of gelatinase secretion after 24 h of incubation of keratinocytes with boron or manganese, regardless of concentration. Gelatinase secretion was not associated with keratinocyte proliferation induced by trace elements. Thus, our results suggest that boron and manganese could play a role in the clinical efficiency of thermal water on wound healing.     

Induction of matrix metalloproteinase-9 in keratinocytes by histamine

Mast cells and their mediator, histamine, are well-recognized effectors in immediate allergy, but their role in tissue remodeling and chronic inflammation has also been the focus of intense research during the past decade. Gschwandtner et al. (2008, this issue) report that histamine can induce MMP-9 expression in keratinocytes, promote collagen type IV degradation in the basement membrane, and stimulate keratinocytes, leading to increased T-cell transmigration through an artificial basement membrane.     

Induction of matrix metalloproteinase-9 secretion from human keratinocytes in culture by ultraviolet B irradiation

BACKGROUND: Matrix metalloproteinases (MMPs) are known as important enzymes involved in tissue metabolism. Among them, MMP-2 and MMP-9 are termed gelatinases, but their specific roles in vivo are still unknown, including their expression patterns following ultraviolet (UV) irradiation. OBJECTIVE: To elucidate the effects of UV irradiation on the skin, we analyzed the expression of MMP-2 and MMP-9 by primary human keratinocytes in culture. METHODS: We evaluated the enzymatic functions of MMP-2 and MMP-9 by gelatin-zymography, and of MMP-9 expression by immunofluorescence, using cultured keratinocytes after UV irradiation. RESULTS: The secretion of MMP-2 (72 kDa) remained at low levels under all conditions examined. Although MMP-9 (92 kDa) secretion was not induced by UVA, it was stimulated by UVB irradiation in a dose-dependent manner. In addition, an enzyme-linked immunosorbent assay showed the tendency to increase for the involucrin expression following UVB exposure. Cell viability was decreased by UVB irradiation in contrast to the induction of MMP-9 and involucrin. CONCLUSION: These results suggest that the induction of MMP-9 secretion is related to the inflammation including apoptosis of keratinocytes resulting from UVB irradiation.     

骨桥蛋白及基质金属蛋白酶—9在恶性黑素瘤中的表达

目的 探讨骨桥蛋白及基质金属蛋白酶-9(MMP-9)在人恶性黑素瘤中的表达及意义。方法 采用免疫组化SP法检测23例原发性恶性黑素瘤、17例转移性恶性黑素瘤及20例色素痣中骨桥蛋白和MMP-9的表达。结果 40例恶性黑素瘤中骨桥蛋白及MMP-9表达的阳性率分别为87.5%、75.0%;20例色素痣中的阳性表达率分别为15.0%、10.0%。骨桥蛋白及MMP-9在恶性黑素瘤中阳性表达率明显高于色素痣,差异具有统计学意义(P<0.05)。骨桥蛋白和MMP-9的表达与年龄、性别、发病部位、是否淋巴结转移等因素均无关(P>0.05)。40例恶性黑素瘤中,骨桥蛋白与MMP-9均表达29例,均不表达4例。结论 骨桥蛋白及MMP-9在人恶性黑素瘤中高表达,但与淋巴结转移无相关性。     

Evaluation of heparanase and matrix metalloproteinase-9 in patients with cutaneous malignant melanoma

Elevated heparanase and matrix metalloproteinase (MMP)-9, frequently found in human cancer, is a major cause of degradation of the extracellular matrix (ECM) and basement membrane (BM), thus facilitating tumor cell migration and invasion. Although a lot of work has been done, the role of heparanase and MMP-9 has not been delineated in skin cancer progression. The purpose of this study was to do such an exploration. To investigate the role of heparanase and MMP-9 in cutaneous malignant melanoma (CMM) development, we performed immunohistochemical analysis to detect the alternation of these two factors in paraffin-embedded biopsy specimens of normal skin, junctional nevi and CMM. It is interesting to note that the expression profile of heparanase and MMP-9 was similar. Contrary to negative staining in normal skin, overexpression of heparanase and cytoplasmic MMP-9 was observed in as many as 70% of CMM, whereas only 10% of the junctional nevi exhibited faint staining (P = 0.0005, P = 0.0000). Considering the lymph node (LN) metastasis, the expression of the two factors is significantly higher in LN-positive lesions than that in LN-negative lesions (P = 0.0295, P = 0.0013). Meanwhile, there was positive correlation between the expression of MMP-9 and heparanase (r = 0.689, P = 0.003). The first expression of MMP-9 and heparanase occurs at benign lesions. However, the significantly increased expression in advanced CMM stages, particularly in LN-positive metastasis lesions, might synergistically contribute to degradation of ECM and BM, therefore promoting carcinogenesis and metastasis.     

MV3恶性黑素瘤皮肤体外模型中基质金属蛋白酶-2、9的表达

目的 探讨体外构建的MV3恶性黑素瘤皮肤模型中基质金属蛋白酶-2（MMP-2）和基质金属蛋白酶-9（MMP-9）的表达及意义。方法 用MV3恶性黑素瘤细胞接种至去表皮的真皮（DED）上,采用液下和空气－液面培养相结合的方式进行组织培养,2周后取培养的黑素瘤组织切片分别做HE染色及MMP-2、9免疫组化染色。同时用RT-PCR对培养组织进行MMP-2、9表达检测。结果 HE染色显示MV3黑素瘤细胞在DED表面呈条带状生长,在凹陷处聚集生长,在DED残留的皮肤附属器管腔中形成大小不等的团、灶;免疫组化染色显示在MV3黑素瘤细胞生长的DED表层及DED附属器管壁基底膜处MMP-2、9均呈阳性表达;RT-PCR检测进一步证实体外构建的恶性黑素瘤组织表达MMP-2和MMP-9,而未接种MV3黑素瘤细胞的DED组织无MMP-2、9表达。结论 体外构建的恶性黑素瘤皮肤模型中瘤细胞侵入到真皮,表达MMP-2、9,为进一步研究黑素瘤的侵袭与转移提供实验依据。     

Matrilysin-2 (matrix metalloproteinase-26) is upregulated in keratinocytes during wound repair and early skin carcinogenesis

Matrilysin-2 (matrix metalloproteinase (MMP)-26) is a small protein of the MMP family expressed in some epithelial carcinomas and normal tissues. We studied its role in benign skin disorders characterized by epithelial proliferation, in wound repair, skin cancer, and regulation in keratinocyte (KC) cultures. MMP-26 is expressed by laminin-5-positive KC in the migrating area during wound repair, in benign skin disorders characterized by inflammation and microdisruptions of basement membrane, but in intact skin only in hair follicles. It was detected in occasional atypical KC in pre-malignant lesions but not in basal cell cancer islands. Although MMP-26 was expressed in grades I and II squamous cell cancers (SCC), it was not present in dedifferentiated grade III tumors. MMP-26 was neither co-expressed with its close homologue matrilysin-1 nor with the proliferation marker Ki-67. But in tissue samples it either co-localized or was detected in adjacent cells of same regions with the tumor suppressor p16. In KC and HaCaT cell cultures, 12-phorbol-13-myristate-acetate, epidermal growth factor, tumor necrosis factor-alpha, transforming growth factor-beta1, interleukin-1 (IL-1)beta, IL-6, insulin-like growth factor, gamma-IFN, retinoic acid, dexamethasone, four matrices or ras-transformation were unable to upregulate MMP-26 expression. The expression pattern of MMP-26 suggests that it may be upregulated in basal KC even without tumorigenesis because of altered cell-matrix interactions and inflammation and, unlike most MMP, becomes downregulated during histological dedifferentiation of SCC. Thus, lack of MMP-26 in SCC could be a marker of aggressive growth.     

Release and activation of matrix metalloproteinase-9 during in vitro mechanical compression in hypertrophic scars

OBJECTIVE: To investigate induction of matrix metalloproteinases (MMPs) during mechanical compression of hypertrophic scars. Mechanical pressure blocks hypertrophy inducted on extracellular matrix in scars by a mechanism that involves MMP-2 (gelatinase A) and MMP-9 (gelatinase B). DESIGN: We assayed conditioned media obtained from normotrophic and hypertrophic scars during 24 hours of in vitro mechanical compression using gelatin zymography. SETTING: Scars from various areas of the bodies of hospitalized patients. PATIENTS: We obtained 3 normotrophic and 7 hypertrophic biopsy specimens from 10 patients (5 men and 5 women). INTERVENTION: In vitro compression at a pressure of 35 mm Hg/cm(2) for 24 hours. MAIN OUTCOME MEASURES: Vitality of scars was analyzed by means of lactic dehydrogenase test; medium samples were collected for zymographic analysis of MMP activity. RESULTS: We found MMP-2 in basal (uncompressed) samples from normotrophic and hypertrophic scars. Mechanical compression induced MMP-9 release and activation (range, 86.7%-78.7%) in hypertrophic scars after 4 hours. CONCLUSION: Production, release, and activation of MMP-9 in hypertrophic scars could be an effector mechanism responsible for hypertrophy regression induced by mechanical compression.     

Vitamin C recycling is enhanced in the adaptive response to leptin-induced oxidative stress in keratinocytes

Leptin acts on energy metabolism and plays a role in skin repair and in the modulation of cellular redox balance as well. Here, we investigated the effects of leptin on the redox homeostasis in keratinocytes, by evaluating reactive oxygen species (ROS) generation, glutathione content, antioxidant enzymes, activating protein 1 (AP-1) activity, and expression of AP-1-dependent, differentiation-specific genes. We also evaluated the systems involved in the maintenance of a positive ascorbate/dehydroascorbate ratio, i.e., transport and recycling. Leptin altered the keratinocyte redox state, as evident by enhanced ROS generation, oxidized/reduced glutathione ratio, and AP-1 activity. Still, this phenomenon was temporary. Indeed, we found an adaptive response, as demonstrated by an early induction of catalase and a late induction of specific dehydroascorbate reductase activities. In particular, leptin-treated cells showed an increased ability to reduce dehydroascorbate, both in a NADH, lipoic acid- and in a NADPH, thioredoxin-dependent manner. Our results show that leptin may induce adaptation to oxidative stress in skin, leading to an improved vitamin C homeostasis.     

Differential response of normal human epidermal keratinocytes and HaCaT cells to hydrogen peroxide-induced oxidative stress

Background. Normal human epidermal keratinocytes (NHEKs) and HaCaT cells are the most common models used to study the effects of various factors on skin cells. These cell lines share some common characteristics, but little is known about their differences in handling hydrogen peroxide (H₂O₂)‐induced oxidative stress. Aim. To investigate the differential response of NHEKs and HaCaT cells to H₂O₂‐induced oxidative stress. Methods. We examined differences in NHEKs and HaCaT cells after H₂O₂ treatment, assessing changes in cell viability; levels of intracellular reactive oxygen species (ROS), superoxide dismutase (SOD) and caspase‐3/7; percentage of cells arrested in G1 phase; number of senescence‐associated β‐galactosidase (SA‐β‐Gal)‐positive cells and; expression of senescence‐related protein Klotho. Results. The viability of NHEKs and HaCaT cells decreased in a concentration‐dependent and time‐dependent manner after exposure to H₂O₂. The inhibitory effect of 150 μmol/L H₂O₂ on cell viability was greater in HaCaT cells than in NHEKs (P < 0.05). Levels of ROS and caspase‐3/7, and the percentage of cells arrested in G1 phase, were higher in HaCaT cells than in NHEKs, whereas intracellular SOD was higher in NHEKs than in HaCaT cells after exposure to 150 μmol/L H₂O₂ (P < 0.05). SA‐β‐Gal positive cells increased significantly in NHEKs after treatment with H₂O₂ (P < 0.05). Klotho was significantly downregulated in both NHEKs and HaCaT cells after H₂O₂ treatment, but no SA‐β‐Gal‐positive HaCaT cells were seen, even after treatment with H₂O₂. Conclusions. Normal human epidermal keratinocytes are more resistant than HaCaT cells to H₂O₂‐induced oxidative stress. HaCaT cells have senescence phenotypes, but do not express β‐Gal.     

MMP-2和MMP-9在多发性肌炎/皮肌炎患者单一核细胞中的表达

目的　探讨基质金属蛋白酶MMP-2和MMP-9 与多发性肌炎/皮肌炎（PM/DM）的关系。方法　采用实时荧光定量PCR和Western印迹方法分别检测PM/DM患者外周血单一核细胞（PMBC）中MMP-2和MMP-9的mRNA和蛋白表达水平。结果　MMP-2、MMP-9在PM/DM患者外周血PMBC中的mRNA表达水平明显高于正常人对照组,其中MMP-2为正常人对照组的2.41倍、MMP-9则为1.66倍。Western印迹检测发现MMP-2、MMP-9在PM / DM 患者外周血PMBC中的蛋白表达水平也明显高于正常人对照组,与其mRNA表达结果一致。结论　基质金属蛋白酶MMP-2和MMP-9可能在PM/DM的发生中起作用。     

Increased matrix metalloproteinase-9 (MMP-9) activity observed in chronic wound fluid is related to the clinical severity of the ulcer

BACKGROUND: The pathology of chronic wounds is often characterized by elevated levels of proinflammatory cytokines [e.g. tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta], proteases [e.g. matrix metalloproteinases (MMPs)] and neutrophil elastase. MMPs specifically have been implicated by a number of studies as the major protease family responsible for the degradation of key factors critical to the ulcer's ability to heal. OBJECTIVES: To assess individual MMPs in chronic wound fluid (CWF) in order to develop improved treatments for chronic ulcers. METHODS: Collagen type I and IV zymography, immunoprecipitation followed by a substrate activity assay, and an indirect enzyme-linked immunosorbent assay were all used to analyse MMP levels in CWF. RESULTS: Our studies demonstrate that there is excessive protease activity in CWF compared with both human serum and acute wound fluid (AWF), which can be specifically attributed to MMPs as determined through a MMP-inhibitor study. Multiple MMPs were immunoprecipitated from the CWF samples and MMP-9 was identified as the predominant protease in CWF, with significantly elevated activity levels in CWF compared with AWF. In addition, the clinical status of the ulcer is directly associated with the amounts of MMP-9 present in the wound fluid. Therefore, this study suggests that higher levels of MMP-9 in chronic wound fluid correlate with a clinically worse wound. CONCLUSIONS: In view of these results, it is hypothesized that a specific inhibitor of MMP-9 could potentially be more therapeutically effective than general MMP inhibitors in modulating chronic ulcers towards a healing state.     

热休克对HaCaT细胞基质金属蛋白酶1和9表达的影响

目的:观察热休克对体外培养的人角质形成细胞系HaCaT细胞表达基质金属蛋白酶1(MMP-1)和9(MMP-9)mRNA以及蛋白质的影响,以阐明环境因素中的热效应在皮肤光老化中所起的作用。方法:将体外培养的人HaCaT细胞分别置于42℃、44℃或46℃水浴箱中30min,然后恢复37℃,5%CO2恒温培养,分别于8h以及48h收集标本,以RT-PCR以及Westernblot方法检测MMP-1和MMP-9的mRNA及蛋白表达。结果:热休克刺激HaCaT细胞8h后MMP-1和MMP-9的mRNA表达温度依赖性上升,热休克刺激48h后HaCaT细胞培养上清中的MMP-1和MMP-9的蛋白表达水平也增高。结论:MMP-1和MMP-9表达过度可能在皮肤光老化的发生及发展中起重要作用。     

Immunohistochemical expression of matrix metalloproteinase-2 and -9 in melanocytic nevi is altered by ultraviolet B

BACKGROUND: Ultraviolet B (UVB) radiation can cause morphological and biological alterations similar to those of melanoma in situ in irradiated melanocytic nevi. matrix metalloproteinase (MMP-2) and -9 appear to be involved with tumour invasion, the formation of metastases and neoangiogenesis in melanomas. The effects of UVB on the immunohistochemical expression of gelatinases in different cell types in melanocytic nevi have not been completely studied. PURPOSE: Evaluate the effects of UVB on the immunohistochemical expression of MMP-2 and -9 in different cell types in melanocytic nevi. METHODS: Forty-two melanocytic nevi had one half irradiated with 2 MEDs of UVB and were excised 1 week later. Three different observers compared the intensity of the immunohistochemical expression of gelatinases in keratinocytes, melanocytes, endothelial cells and fibroblasts on the irradiated (IS) and non-irradiated sides (NIS). RESULTS: All the cell types showed an increase in the expression of MMP-2 on the IS, especially the epidermal melanocytes. No significant increase in the expression of MMP-9 in keratinocytes was detected on the IS; significant increase was observed in all the remaining cells. CONCLUSIONS: One single irradiation of UVB increases the immunohistochemical expression of gelatinases in almost all evaluated cell lines, with the exception of MMP-9 in keratinocytes.     

Platelet lysate modulates MMP-2 and MMP-9 expression, matrix deposition and cell-to-matrix adhesion in keratinocytes and fibroblasts

Cell-matrix interactions are an essential element of wound healing, while platelet derivatives are used in clinical settings for the treatment of chronic wounds. We used a platelet lysate (PL), which had been previously shown to accelerate in vitro the wounding of HaCaT keratinocytes and fibroblasts (J Cell Mol Med, 13, 2009, 2030; Br J Dermatol, 159, 2008, 537), to study the modulation of MMP-2 and MMP-9 collagenase expression, collagen type I and III production and syndecan-4 expression and rearrangement in these cells. Zymography and Western blot analyses showed that exposure to 20% (v/v) PL for 24 h induced an apparently ERK1/2- and p38-dependent, NF-kappaB-independent, translational upregulation of MMP-9 in HaCaT, while HaCaT MMP-2 and fibroblast collagenases were almost unaffected. The use of in-cell ELISA showed that PL induced an increase in the collagen III production of fibroblasts. In-cell ELISA and immunofluorescence microscopy revealed an increase in the expression of syndecan-4 and its rearrangement to form focal adhesions in both cell types after PL exposure. Taken together, data indicate that PL promotes keratinocyte epithelialization and regulates fibroblast matrix deposition, thus providing a molecular basis for the ability of this platelet derivative to heal severe and problematic wounds without leading to heavy scarring and keloid formation.     

Human keratinocytes respond to osmotic stress by p38 map kinase regulated induction of HSP70 and HSP27.

Human skin is exposed to an environment that varies in humidity from 100 to 0%, leading to seasonal variations in the condition of the skin. Exposure to a low humidity environment creates an osmotic gradient across the stratum corneum, which is known to modulate cutaneous barrier function. Heat shock proteins protect against stress-induced destabilization of proteins. We investigated whether osmotic shock (sorbitol) induced a heat shock protein response in normal human keratinocytes, and used heat shock as a positive control. Both heat shock and osmotic stress (200 and 300 mM sorbitol) clearly induced heat shock proteins 70 and 27 mRNA levels. The induction of heat shock protein 70 mRNA levels by osmotic stress peaked at 16 h and persisted until 24 h, whereas upregulation of heat shock protein 70 mRNA levels by heat peaked at 2 h and returned to baseline levels by 6 h. Sorbitol also increased heat shock protein 70 levels in a concentration-dependent manner. The kinetics of heat shock protein 27 mRNA induction by osmotic stress and heat were similar with peak induction at 6 h. The mitogen activated protein kinase family of proteins plays an important part in the coordination of gene responses to various stress conditions. We have demonstrated that the p38 mitogen activated protein kinase was strongly activated by 200 mM and 300 mM sorbitol. The specific p38 mitogen activated protein kinase inhibitor PD169316 almost completely blocked heat shock protein 70 mRNA induction by 200 mM and 300 mM sorbitol and completely suppressed heat shock protein 27 mRNA induction with 200 mM sorbitol. PD169316 also counteracted upregulation of heat shock protein 70 levels by sorbitol. These data indicate that keratinocytes respond to osmotic stress by p38 mitogen activated protein kinase regulated induction of heat shock proteins. This molecular pathway may be relevant for the mechanisms regulating the response of human skin to variations in environmental humidity.     

G2A plays proinflammatory roles in human keratinocytes under oxidative stress as a receptor for 9-hydroxyoctadecadienoic acid

G2A is a stress-inducible G protein-coupled receptor for oxidized free fatty acids, such as 9-hydroxyoctadecadienoic acid (HODE). As skin is routinely and pathologically exposed to many oxidative stresses such as UV radiation, chemical agents, and inflammation that might induce both G2A expression and production of G2A ligands, we examined G2A function in human keratinocytes. G2A was expressed in human epidermis, normal human epidermal keratinocytes (NHEK), and an immortalized human keratinocyte cell line (HaCaT). 9(S)-HODE evoked intracellular calcium mobilization and secretion of cytokines, including IL-6, IL-8, and GM-CSF in NHEK cells. These responses became prominent in HaCaT cells by overexpression of G2A. 9(S)-HODE inhibited proliferation of NHEK cells by suppressing DNA synthesis and arresting the cell cycle in the G0/1-phase. On the other hand, 13(S)-HODE, another major oxidative product from linoleate, showed little or no effect on either cytokine secretion or on proliferation in NHEK cells. A small interfering RNA designed to downregulate G2A caused suppression of 9(S)-HODE-induced inhibitory effects on proliferation of NHEK cells. UVB and H(2)O(2) induced G2A expression and caused oxidation of linoleate to produce 9-HODE in HaCaT cells. These results suggest that 9-HODE-G2A signaling plays proinflammatory roles in skin under oxidative conditions.     

Endothelial protein C receptor and protease-activated receptor-1 mediate induction of a wound-healing phenotype in human keratinocytes by activated protein C

Activated protein C (APC) is a natural anticoagulant and inhibitor of inflammation that can stimulate keratinocyte wound repair in vitro and promote wound healing in vivo. The signaling mechanisms, however, are unknown and a keratinocyte receptor for APC has not been identified. Here, we show that cultured human keratinocytes from neonatal foreskins express the endothelial protein C receptor (EPCR). EPCR was also strongly expressed by lower epidermal layers of neonatal foreskin as determined by immunohistochemistry. In cultured keratinocytes, EPCR expression was upregulated by the addition of APC and inhibited by tumor necrosis factor-alpha. Addition of APC stimulated cell proliferation, production of matrix metalloproteinase-2, activation of ERK and p38 kinase signaling pathways, and expression of protease-activated receptor (PAR)-1. A monoclonal antibody, RCR252, which blocks APC binding to EPCR, or a blocking antibody to PAR-1, abolished APC's effects on keratinocytes. In summary, this study demonstrates that EPCR, a major receptor of protein C pathway, is expressed by human keratinocytes, and facilitates APC's function on keratinocytes via activation of PAR-1 pathway. Our findings highlight a possible new role for the protein C pathway in skin physiology and help elucidate the mechanisms of action by which APC promotes wound healing.