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.     

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.     

UVA联合UVB照射对原代人角质形成细胞增殖的影响

目的 探讨95％长波紫外线(UVA)联合5％中波紫外线(UVB)照射对原代人表皮角质形成细胞(HEK)增殖的影响,为构建日光对人工皮肤光损伤模型提供实验依据.方法 用总剂量分别为0、2.5、5、10、20、30、40、60 J/cm2的紫外线(含95％UVA和5％UVB),分别照射体外培养的HEK,继续培养24 h后,用噻唑蓝(MTr)法检测细胞活力,用SPSS软件计算引起HEK半数损伤时的紫外线照射剂量.结果 8种剂量紫外线照射后,HEK相对抑制率分别为0、1.03％、6.60％、17.28％、31.28％、49.59％、59.67％、70.99％,当总照射剂量≥10 J/cm2时,HEK相对抑制率与对照组相比,差异有统计学意义(F=62.11,P＜0.05);HEK相对抑制率为50％时的紫外线照射总剂量为31.31 J/cm2.结论 随着紫外线照射剂量的加大,HEK增殖活力降低,31.31 J/cm2为HEK半数死亡的照射剂量.     

Mitochondrial dysfunction and cellular stress progression after ultraviolet B irradiation in human keratinocytes

Background: Ultraviolet (UV) radiation is the major environmental harmful factor that affects human skin. UVB radiation is known to be a potent inducer of reactive oxygen species (ROS) production and has also been associated with the generation of nitric oxide (NO), all of which have been implicated in various skin disorders. It is well known that mitochondria can also be affected by UVB, leading to alterations in their membrane structure and permeabilization with cytochrome c release, which consequently affects the cell function. However, the loss of keratinocyte mitochondrial function generated by UVB, as well as its kinetics, has not been characterized completely.
Methods: We evaluated the effect of UVB irradiation on HaCat cells' mitochondrial function, assessed by membrane potential loss and superoxide anion (O₂^•−) production, correlating with apoptosis, p53 expression, ROS levels and NO production, 0, 6, 12, 24 and 48 h post-irradiation.
Results: HaCat cells progressed toward apoptotic cell death as the time post-irradiation increased, with the highest levels found 48 h after irradiation. Increased levels of ROS were observed 6 h after irradiation while high O₂^•− levels and mitochondrial membrane depolarization were detected 12 h post-UVB. Nevertheless, NO production was not significantly increased at any of the evaluated times.
Conclusions: The kinetics of mitochondrial dysfunction after UVB irradiation in human keratinocytes progressed in a time post-irradiation-dependent manner, and they are closely related to cell death. However, there are certain levels of apoptosis, although low, in the absence of mitochondrial alterations. In addition, our data suggest that ROS play a greater role in keratinocyte UVB damage than reactive nitrogen species.     

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.     

Apoptosis induced by ultraviolet B in HPV-immortalized human keratinocytes requires caspase-9 and is death receptor independent

Ultraviolet B (UVB) induces both apoptosis and skin cancer. We found that human keratinocytes (KC) immortalized by Human Papillomavirus (HPV)16 E6/E7 were sensitized to UVB-induced apoptosis, possibly representing a transient regression-prone precancerous stage equivalent to actinic keratosis. To further examine which caspases are apical and essential, we utilized retroviral constructs expressing dominant-negative caspase-9 (caspase-9-DN) or Fas-associated protein with death domain (FADD)-DN as well as caspase inhibitor peptides. Caspase-9-DN and zLEHD-fmk both suppressed caspase-9, -3, and -8 activity after UVB exposure, as well as proteolytic processing of procaspase-3 into its active form, DNA fragmentation factor 45 cleavage, and internucleosomal DNA fragmentation. By contrast, stable expression of FADD-DN in HPV-immortalized KC did not inhibit UVB-induced activation of caspases-9, -3, and -8 nor downstream apoptotic events, although inhibition of caspase-8 with zIETD-fmk attenuated apoptosis. This study indicates that caspase-9 activation is upstream of caspases-3 and -8 and that UVB-induced apoptosis in HPV-immortalized human KC is death receptor (DR) independent and requires both caspase-9 upstream and caspase-8 downstream for maximal apoptosis. These studies further indicate that cell type as well as transformation state determine the sensitivity and mode of cell death (DR vs. mitochondrial apoptotic pathways) in response to UVB and explain the high regression rates of premalignant lesions.     

中波紫外线辐射损伤角质形成细胞的p53信号传导通路研究

目的 探讨中波紫外线(UVB)辐射对角质形成细胞p53信号传导通路的影响.方法 以20、60和120mJ/cm²UVB辐射培养的取自健康儿童包皮的正常人表皮角质形成细胞,应用RT-PCR和免疫印迹方法,分别从mRNA水平和蛋白质水平,检测分析UVB辐射后2h、24h和48h,p53及其下游分子MDM2、p21、Bax和GADD45的表达.结果 不同剂量UVB辐射角质形成细胞后均可见p53表达水平持续性显著升高(P<0.05).低剂量UVB辐射角质形成细胞后,MDM2、p21、GADD45升高不明显(P>0.05),Bax不表达;UVB辐射剂量升至60mJ/cm²后,MDM2于辐射后2h短暂性显著升高,p21和Bax持续性显著升高.结论 UVB辐射激活了角质形成细胞的p53信号传导通路,p53下游分子的表达具有剂量依赖性和时相性.     

西罗莫司对UVB照射HaCaT细胞自噬的影响

目的 探讨西罗莫司能否增加中波紫外线(UVB)照射下HaCaT细胞的自噬水平.方法 0、25、50 mJ/cm2UVB照射HaCaT细胞,单丹(磺)酰戊二胺(MDC)染色法检测自噬.设置空白组、DMSO组、25 mJ/cm2UVB组、25 mJ/cm2 UVB+西罗莫司组、50 mJ/cm2UVB组、50 mJ/cm2 UVB+西罗莫司组共6组,MDC染色法检测自噬,并比较各组自噬水平.结果 结果UVB照射HaCaT细胞后出现了自噬,0、25、50 mJ/cm2照射组自噬体阳性细胞分别为1.07％±1.85％、9.37％±1.14％、16.29％±3.03％,3个剂量间差异有统计学意义;6组自噬体阳性细胞百分率分别为:0.56％±0.79％、1.61％±2.27％、10.00％±0.47％、21.18％±3.03％、15.82％±4.13％、29.45％±1.24％,各组两两比较差异均有统计学意义,且25 mJ/cm2UVB+西罗莫司组、50 mJ/cm2 UVB+西罗莫司组的自噬水平均高于相应UVB组.结论 UVB辐照HaCaT细胞后出现MDC染色自噬体阳性细胞,西罗莫司可能增加UVB照射下HaCaT细胞后的自噬水平.     

Differential phosphorylation of mitogen-activated protein kinase families by epidermal growth factor and ultraviolet B irradiation in SV40-transformed human keratinocytes

SV-40 transformed human keratinocytes (SVHK cells) were stimulated with epidermal growth factor (EGF) and ultraviolet B (UVB) irradiation. Following the stimulation, cell growth, apoptosis, and the activities of mitogen-activated protein (MAP) kinase families were analyzed. EGF (100 ng/ml) increased SVHK cell number compared with control cells cultured in serum-free DMEM medium. The EGF-stimulated cells did not show DNA fragmentation. In contrast, UVB irradiation (40 mJ/cm(2)) markedly decreased viable cell number that was accompanied with DNA fragmentation. EGF stimulated extracellular signal-regulated kinase (ERK) and stress-activated protein kinase/c-Jun N-terminal kinase (JNK). Following the EGF stimulation, phosphorylated ERK and JNK were detected by phospho-p42/44 MAP kinase antibody and phospho-SAPK/JNK antibody, respectively. On the other hand, UVB irradiation stimulated the phosphorylation of p38 and JNK but not of ERK. The stimulation of ERK and JNK induced by EGF was observed earlier than the stimulation of p38 and JNK induced by UVB. PD98059, a specific MAP kinase kinase (MAPKK) 1 (also referred to as MEK1) inhibitor, inhibited EGF-dependent cell proliferation, that was associated with the inhibition of ERK and JNK phosphorylation. In contrast, UVB-induced overall cell death was not significantly affected by PD98059, that inhibited phosphorylation of JNK but not of p38. PD98059, however, significantly augmented UVB-induced cell death earlier time points (30 min--2 h). These results indicate that ERK and JNK are activated following EGF stimulation that might be associated with cell proliferation. On the other hand, UVB-induced apoptosis seems to be mostly associated with the activation of p38. JNK stimulation might provide an anti-apoptotic tonus during the UVB-induced, p38-associated SVHK cell death.     

Differential response of basal keratinocytes in a human skin equivalent to ultraviolet irradiation

Abstract The human skin equivalent (HSE) provides a convenient model system for studying the cellular responses of basal keratinocytes to UV irradiation. HSEs, constructed by overlaying a collagen-fibroblast matrix with epidermal cells, were raised to an air-liquid interface to promote epidermal differentiation. HSEs were exposed to ultraviolet radiation from a 500-W Hot Quartz Hanovia therapeutic sunlamp, at a total dose of 100 J/m². The HSEs were then frozen every 4 h over a 48-h period and cryosectioned. For each time period, the expression of β₁ integrin and cyclin E, p53, or Bcl-2 were quantified using dual immunolocalization. Basal cells expressing β₁ integrin were divided into two subpopulations, denoted β₁ ^high or β₁ ^low. The proportion of β₁ ^high keratinocytes expressing Bcl-2 and cyclin E increased significantly 4 and 8 h, respectively, after exposure to UV; during the subsequent 16 h, this basal cell subpopulation expressed p53. By contrast, significant numbers of β₁ ^low basal keratinocytes expressed p53, but not Bcl-2. These results suggest that β₁ ^high and β₁ ^low populations of basal epidermal cells in HSEs respond differently to UV irradiation. Received: 30 October 1997     

Effects of ultraviolet A and B irradiation on lipid peroxidation and activity of the antioxidant enzymes in keratinocytes in culture.

Human keratinocytes (NCTC 2544) in culture were exposed to various combinations of ultraviolet A (UVA) and UVB irradiation and at 0.5 h postirradiation the level of lipid peroxidation and activities of antioxidant enzymes were measured. The results suggest that UV irradiation is capable of inducing lipid peroxidation reactions, as parameters of which the amount of thiobarbituric acid-reactive material and the number of conjugated diene double bonds were measured. Both UVA and UVB irradiation were also found to affect the activities of antioxidant enzymes. Following UVB irradiation the activity of superoxide dismutase (Cu/Zn form) was decreased, and combination of increasing doses of UVA irradiation to a given dose of UVB irradiation decreased the activity of both catalase and superoxide dismutase. In summary, this study suggests that both UVA and UVB irradiation are capable of inducing lipid peroxidation reactions and an impairment of the enzymic antioxidant system in human keratinocytes in culture.     

The effect of ultraviolet B irradiation on nitric oxide synthase expression in murine keratinocytes

Nitric oxide (NO), which has several physiological functions in skin, is generated by NO synthase (NOS). NOS has at least three isoforms; endothelial NOS (eNOS), brain NOS (bNOS), and inducible NOS (iNOS). Ultraviolet B (UVB) irradiation has been reported to stimulate NO production in skin via induction or activation of NOS, however, the exact mechanism of NOS induction by UVB irradiation remains obscure. In this study, we investigated the direct effect of UVB on the expression of NOS isoforms in murine keratinocytes, and found a significant increase in NO production within 48 h. mRNA and protein expressions of bNOS were both enhanced by UVB irradiation in murine keratinocytes, whereas iNOS mRNA expression was suppressed at 4 and 12 h after UVB irradiation. These results suggest that the enhancement of NO production observed after UVB irradiation in murine keratinocytes may be explained in part by the upregulation of bNOS expression, but not iNOS expression.     

Ultraviolet irradiation increases matrix metalloproteinase-8 protein in human skin in vivo

Humans express three distinct collagenases, MMP-1, MMP-8, and MMP-13, that initiate degradation of fibrillar type I collagen. We have previously reported that ultraviolet irradiation causes increased expression of MMP-1, but not MMP-13, in keratinocytes and fibroblasts in human skin in vivo. We report here that ultraviolet irradiation increases expression of MMP-8 in human skin in vivo. Western analysis revealed that levels of the full-length, 85 kDa proenzyme form of MMP-8 increased significantly within 8 h post ultraviolet irradiation (2 minimal erythema doses). Increased full-length MMP-8 protein was associated with infiltration into the skin of neutrophils, which are the major cell type that expresses MMP-8. Immunofluorescence revealed coexpression of MMP-8 and neutrophil elastase, a marker for neutrophils. Immunohistology demonstrated MMP-8 expression in neutrophils in the papillary dermis between 4 and 8 h post ultraviolet irradiation, and in the epidermis at 24 h post radiation. MMP-8 mRNA expression was not detected in nonirradiated or ultraviolet-irradiated human skin, indicating that increased MMP-8 following ultraviolet irradiation resulted from preexisting MMP-8 protein in infiltrating neutrophils. Pretreatment of skin with the glucocorticoid clobetasol, but not all-trans retinoic acid, significantly blocked ultraviolet-induced increases in MMP-8 protein levels, and neutrophil infiltration. In contrast, all-trans retinoic acid and clobetasol were equally effective in blocking ultraviolet induction of MMP-1 and degradation of collagen in human skin in vivo. Taken together, these data demonstrate that ultraviolet irradiation increases MMP-8 protein, which exists predominantly in a latent form within neutrophils, in human skin in vivo. Although ultraviolet irradiation induces both MMP-1 and MMP-8, ultraviolet-induced collagen degradation is initiated primarily by MMP-1, with little, if any, contribution by MMP-8.     

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。