Expression of matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, and MT1-MMP in skin tumors of human papillomavirus type 8 transgenic mice

Human papillomaviruses (HPV) are small DNA viruses that induce a wide variety of hyperproliferative lesions in cutaneous and mucosal epithelia. It is proposed that HPV is involved in non-melanoma skin cancer development. We have previously shown that HPV8 transgenic mice spontaneously develop papillomatous skin tumors. Histology revealed epidermal hyperplasia, acanthosis and hypergranulosis and in some cases squamous cell carcinomas (SCC). Zymographic and immunoblot analysis of normal skin extracts identified increased amounts of matrix metalloproteinase (MMP)-9, MMP-13 and MT1-MMP in HPV8-positive mice compared with HPV8-negative animals. In situ gelatin zymography of tumor specimens displayed a strong proteolytic activity in papillomas, and SCC putatively attributed to the increased amounts of activated MMP-9 found in tissue extracts. In addition, immunoblot analysis revealed increased amounts of active MMP-13 and MT1-MMP in tumor extracts as compared with control extracts. Immunohistochemical stainings of SCC specimens depicted MMP-13 to be specifically expressed in stromal fibroblasts neighboring the tumor islands, whereas MT1-MMP was detected both in tumor cells and in stromal cells. Taken together, these results implicate a role for MMPs in the development of HPV8-induced cutaneous tumors.     

Lymphocyte infiltration of the skin in transgenic mice carrying the human interleukin-2 gene

Inflammatory lesions of the skin such as erythema, depigmentation and hair loss were observed in C57/BL6(B6) transgenic mice that carried an intact human genomic interleukin-2 gene (gIL-2 transgenic mice). Accumulation of T lymphocytes in the perivascular and periadnexal areas of the dermis was the first change, followed by dermal papillary oedema, which occurred before the development of macroscopic skin lesions. In 3- or 4-week-old transgenic mice with slight erythema and depigmentation of the skin, there was an increase in the number of perivascular lymphocytes accompanied by the diffuse infiltration of neutrophils and monocytes in the damaged skin. These morphological skin changes were not observed in non-transgenic mice, which were bred together with transgenic litter mates. These findings suggest that lymphocyte infiltration of the perivascular space of the skin is a primary event of exogenously introduced human interleukin-2 gene, resulting in secondary cutaneous changes in gIL-2 transgenic mice.This work was presented in part at the 52nd Annual Meeting of the Society for Investigative Dermatology, Seattle, Wash., 1–3 May 1991     

HPV8 early genes modulate differentiation and cell cycle of primary human adult keratinocytes

Human papillomaviruses (HPV) have been associated with the development of non-melanoma skin cancer (NMSC) but the molecular mechanisms of the role of the virus in NMSC development are not clearly understood. Abnormal epithelial differentiation seen in malignant transformation of keratinocytes is associated with changes in keratin expression. The purpose of this study was to investigate the phenotype of primary human adult keratinocytes expressing early genes of HPV8 with specific reference to their differentiation and cell cycle profile to determine whether early genes of HPV8 lead to changes that are consistent with transformation. The expression of HPV8 early genes either individually or simultaneously caused distinct changes in the keratinocyte morphology and induced an abnormal keratin expression pattern, that included simple epithelial (K8, K18, K19), hyperproliferation-specific (K6, K16), basal-specific (K14, K15) and differentiation-specific (K1, K10) keratins. Our results indicate that expression of HPV8 early genes disrupts the normal keratin expression pattern in vitro. Expression of HPV8-E7 alone caused polyploidy that was associated with decreased expression of p21 and pRb. Expression of individual genes or in combination differentially influenced cell morphology and cell cycle distribution which might be important in HPV8-induced keratinocyte transformation.     

Skin tumor formation in human papillomavirus 8 transgenic mice is associated with a deregulation of oncogenic miRNAs and their tumor suppressive targets

Background

Dysregulation of microRNA (miRNA) expression is regularly found in various types of cancer and contributes to tumorigenic processes. However, little is known about miRNA expression in non-melanoma skin cancer in which a pathogenic role of beta human papillomaviruses (HPV) is discussed. A carcinogenic potential of beta HPV8 could be demonstrated in a transgenic mouse model, expressing all early genes of HPV8 (HPV8-CER). A single UVA/B-dose induced oncogene expression and led to papilloma growth within three weeks.

Objective

Expression of miRNAs and their targets during HPV8-mediated tumor formation in mice.

Methods

Skin of untreated or UV-irradiated wild-type and HPV8-CER mice was analyzed for miRNA expression and localization by qPCR and in situ hybridization. MiRNA target protein expression was analyzed by immunohistochemical staining.

Results

Early steps in skin tumor formation in HPV8-CER mice were associated with an upregulation of the oncogenic miRNA-17-5p, -21 and -106a and a downregulation of the tumor-suppressive miRNA-155 and -206, which could be demonstrated by qPCR and in situ hybridization. The respective targets of miRNA-21 and -106a, the tumor suppressors PTEN, PDCD4 and Rb with their pivotal role in cell cycle regulation, apoptosis and proliferation were found to be downregulated.

Conclusion

This is the first report demonstrating that a cutaneous HPV type deregulates the expression of miRNAs. These deregulations are closely related to the UV-induced upregulation of HPV8 oncogene levels, which suggest a direct or indirect HPV8-specific effect on miRNA expression. These data presume that HPV8 interferes with the miRNA mediated gene regulation to induce tumorigenesis.     

The effect of proteolytic enzymes on hair follicles of transgenic mice expressing the lac Z-protein in cells of the bulge region

OBJECTIVE: To study the effects of proteolytic enzymes on mice hair follicles, particularly on cells of the bulge area regarded as follicle stem cells. BACKGROUND: Previous application by iontophoresis of proteolytic enzymes on guinea pig skin resulted in degenerative effects on hair follicles and the hypothesis was proposed that some of the affected cells could be stem cells. METHODS: To mark putative stem cells transgenic mice were produced carrying the lac-Z gene fused to the Upstream Regulatory Region (URR) of Human Papilloma Virus 11 (HPV11), as they express this gene specifically in the cells of the bulge area. Chymotrypsin and papain were applied on skin by iontophoresis, trypsin in the form of liposomes. RESULTS: Enzyme application, both by electrophoresis and as liposomes, led to intense degenerative effects of the hair follicle, such as detachment of the inner root sheath, cystic dilation of the hair shaft and presence of epithelial cells within the lumen. Some of these cells represent hair follicle stem cells expressing beta-galactosidase (beta-gal), having been detached from the bulge area as a result of enzyme treatment, implying impairment of their function.     

The HPV16 E6 oncoprotein and UVB irradiation inhibit the tumor suppressor TGFβ pathway in the epidermis of the K14E6 transgenic mouse

High‐risk human papillomaviruses (HR‐HPVs) are the causative agents of cervical cancer, and they are also associated with a subset of head and neck squamous cell carcinomas. In addition, HPVs have also been postulated in the development of non‐melanoma skin cancers (NMSC). In these cancers, the oncogene E6 is best known for its ability to inactivate the tumor suppressor p53 protein. Interestingly, in transgenic mice for HPV16 E6 (K14E6), it was reported that E6 alone induced epithelial hyperplasia and delay in differentiation in skin epidermis independently of p53 inactivation. Transforming growth factor β (TGFβ) is an important regulator of cell growth/differentiation and apoptosis, and this pathway is often lost during tumorigenesis. Ultraviolet radiation B (UVB) exposure activates diverse cellular responses, including DNA damage and apoptosis. In this study, we investigated whether the E6 oncogene alone or in combination with UVB dysregulate some components of the TGFβ pathway in the epidermis of K14E6 mice. We used 8‐day‐old K14E6 and non‐transgenic mice irradiated and unirradiated with a single dose of UVB. We found that the E6 oncogene and UVB irradiation impair the TGFβ pathway in epidermis of K14E6 mice by downregulation of the TGFβ type II receptor (TβRII). This loss of TβRII prevents downstream activation of Smad2 and target genes as p15, an important regulator of cell cycle progression. In summary, the TGFβ signalling in cells of the epidermis is downregulated in our mouse model by both the E6 oncoprotein and the UVB irradiation.     

VEGF expression in skin warts. Relevance to angiogenesis and vasodilation

Abstract Verrucae vulgaris (skin warts) are benign proliferative lesions which are generally associated with human papillomavirus type 2 (HPV-2) infection. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen able to induce angiogenesis and vasodilation. Our previous findings indicate that these two processes take place during the formation of skin warts. The purpose of this study was to determine whether VEGF expression in these lesions was associated with HPV infection, angiogenesis or vasodilation. To this end, paraffin-embedded specimens of skin warts which were either negative for HPV-1, -2, -3 and -4 (HPV^–; n = 18), or positive for HPV-2 (HPV⁺; n = 21) were compared with histologically normal perilesional skin (n = 13). Serial sections were stained with antibodies to von Willebrand Factor (vWF) and to VEGF. Vascularity was quantified by point counting vWF-positive blood vessels. Small and large vessels were quantified separately, using a cut-off value of 50 μm diameter. VEGF expression in the epidermis was estimated by consensus of two independent observers according to three indices: (1) percentage of cells stained, (2) intensity of the staining, and (3) product of area and intensity (final score). Results were analysed by nonparametric tests. Similar levels of VEGF were found in specimens of normal skin, HPV^– and HPV⁺ warts, irrespective of the index used. There was no significant correlation between VEGF expression and vascularity values for either small or large vessels. These results indicate that, on its own, VEGF expression is not associated with angiogenesis, vasodilation or HPV infection in skin warts. The presence of VEGF in normal skin suggests that it may play a role in tissue homeostasis. Received: 23 May 2000 / Revised: 21 August 2000 / Accepted: 9 February 2001     

尖锐湿疣及鲍恩样丘疹病皮损中高危型人乳头瘤病毒感染与端粒酶表达的关系

目的:研究尖锐湿疣(CA)与鲍恩样丘疹病(BP)皮损中高危型人乳头瘤病毒(HPV)感染与端粒酶表达的关系。方法:采用PCR方法检测HPV类型,免疫组化方法检测CA和BP皮损中人类端粒酶反转录酶(hTERT)的表达。结果:CA和BP皮损中hTERT表达均比正常对照组高(P均<0.01),BP又较CA表达高(P<0.01)。CAHPV16阳性组hTERT表达较HPV16阴性组高,其差异有统计学意义(P<0.05);BP不同HPV型之间hTERT表达的结果与CA相同;以上各组均比正常对照组表达强度高(P<0.05)。结论:非恶性增殖性疾病中亦有端粒酶不同程度的表达;以HPV16为代表的高危型HPV感染与端粒酶表达有相关性。     

The human papillomavirus type 8 E2 gene encodes a transforming activity sufficient for skin tumor formation in transgenic mice

Infections with beta-genus human papillomaviruses (HPVs) have been associated with nonmelanoma skin cancers, particularly in immunocompromised patients and individuals with a rare genetic disease, epidermodysplasia verruciformis (EV). Using a transgenic mouse model, Herbert Pfister's group determined that expression of the HPV8 E2 gene results in skin cancer development and that this process is greatly accelerated by UV irradiation (Pfefferle et al., 2008, this issue).     

Action spectra for 8-methoxypsoralen and 3-carbethoxypsoralen in skin fibroblasts in vitro

Summary 8-methoxypsoralen (8-MOP) and 3-Carbethoxypsoralen (3-CPs) are known photoreagents which have been employed in the treatment of psoriasis. Using skin fibroblasts grown in vitro we have determined the action spectra of both compounds in the long ultraviolet range. Narrow-band interference filters were applied to a Xenon lamp. Reduction in methyl-³H-thymidine (³H-TdR) uptake served as a parameter for photoinhibited cells. The results show a linear increate of 8-MOP-mediated photoinhibition with increasing wavelengths; 3-CPs showed comperatively weak inhibitory rates at the lower wavelength range (349–365 nm). Possibly this is due to the formation of 3-CPs photoproducts which are unreactive with DNA. Compared to 8-MOP, the monofunctional photoreagent 3-CPs is a weak photoinhibitor.This work was supported by Stiftung Volkswagenwerk     

昆明山海棠对人单核细胞TNFα、IL－8及基因表达的影响

应用体外细胞培养方法，ELISA及RT－PCR技术，观察昆明山海棠对健康人外周血单核细胞IL－8，TNFα释放及IL－8 mRNA表达的影响。结果表明昆明山海棠（0．2mg/mL-1mg/mL)可抑制脂多糖（LPS）诱导的外周血单核细胞IL－8，TNFα释放及IL－8 mRNA表达，提示昆明山海棠可从转录水平抑制细胞因子表达，从而实现其抗炎机制。     

宫颈上皮内瘤变患者高危型人乳头状瘤病毒感染与吲哚胺2,3-二氧酶表达的关系

目的：研究高危型人乳头状瘤病毒与吲哚胺2,3-二氧酶在宫颈上皮内瘤变组织中表达情况.方法：采用第二代杂交捕获法检测高危型HPV,免疫组化法检测吲哚胺2,3-二氧酶表达.结果：高危型HPV感染与吲哚胺2,3-二氧酶同时阳性的患者占62.91%（95例/151例）,同时阴性的患者占21.85%（33例/151例）.高危型HPV感染阳性的患者宫颈组织中吲哚胺2,3-二氧酶阳性表达率显著高于高危型HPV感染阴性患者（χ2=62.1833,P〈0.01）.高危型HPV与吲哚胺2,3-二氧酶表达有相关性（r=0.9611,P〈0.05）.结论：高危型HPV联合吲哚胺2,3-二氧酶在宫颈上皮内瘤变的疾病发生、发展过程中起到重要作用.     

脓疱性银屑病患者外周血IL-8水平及其在皮损中的表达

许多学者发现,IL-8与银屑病发病有密切关系,脓疱性银屑病皮损中有比寻常性银屑病皮损中更多的中性粒细胞浸润和IL-8持续升高.2004年7月至2006年2月,我们采用双抗体夹心法及免疫组化技术检测脓疱性银屑病、寻常性银屑病及正常人对照组外周血IL-8水平及皮损处IL-8的表达情况.     

Enhanced expression of IL-8 in normal human keratinocytes and human keratinocyte cell line HaCaT in vitro after stimulation with contact sensitizers., tolerogens and irritants

Abstract To investigate the interleukin-8 production of keratinocytes after stimulation in vitro we have used various agents: (i) contact sensi-tizer (2,4-dinitrofiuorobenzene, 3-n-penladecylcatechol); (ii) tolerogen (5-methyl-3-n-pentadecylcatechol); (iii) irritant (sodium lauryl sulfate). Interleukin-8 gene expression was assessed by northern blot hybridization of the total cytoplasmic RNA extracted from subconfluent normal human keratinocyte cultures and the keratinocyte cell line HaCaT using a radiolabeled DNA probe specific for human interleukin-8. Intcrleukin-8 gene expression was markedly increased upon in vitro stimulation after 1-6 h with contact sensitizers, tolerogen and the irritant. In contrast, in-terlcukin-8 production was not detectable in unstimulatcd normal human keratinocytes or the HaCaT keratinocyte cell line. These results suggest that the induction and production of interleukin-8 is a response to nonspecific stimuli and may play a critical role in the early response to immuno-genic or inflammatory signals in man.     

The expression of interleukin-8 receptor in untreated and treated psoriasis

The expression of IL-8 in psoriasis has been clearly shown with the use of immunocytochemical, RT-PCR and in situ hybridization methods. The presence of its ligand, the IL-8 receptor, has been demonstrated by the RT-PCR technique. We report here a study of the expression of both IL-8 type A and B receptors by immunohistochemical techniques, using one polyclonal and four monoclonal antibodies. By this technique, we found that the neutrophilic granulocytes express the IL-8 type A receptor, whereas the IL-8 type B receptor was present on the keratinocytes. The type B receptor on the keratinocytes was localized in the suprabasal layers of the epidermis. Following therapy, the expression of the IL-8 type B receptor on the keratinocytes was reduced. This could suggest that IL-8 in psoriasis is involved in the disturbed differentiation rather than in proliferation, probably via an autocrine loop. Received: 18 October 1996     

P53,Fas,Bcl-2蛋白在尖锐湿疣组织中的检出及其与HPV感染的相关性

目的　探讨P5 3 ,Fas ,Bcl 2蛋白在尖锐湿疣 (CA)组织中的检出情况及其与人乳头瘤病毒 (HPV)感染的关系。方法　采用标准免疫组化法检测了 2 4例CA、46例宫颈癌和 2 8例正常宫颈组织P5 3 ,Fas ,Bcl 2蛋白 ,用PCR技术对各组织标本的HPV DNA进行检测和 6,11,16,18分型。结果　P5 3 ,Fas ,Bcl 2蛋白在CA组的阳性率显著高于正常对照组 (P <0 .0 0 1) ;CA组和宫颈癌组之间差异无显著性 (P >0 .0 5 )。综合分析 ,蛋白阳性率与HPV感染率密切相关 (P <0 .0 5 ) ,但CA组或宫颈癌组的蛋白阳性率与HPV 6/11型或 16/18型的检出与否无统计学意义 (P >0 .0 5 )。结论　P5 3 ,Fas ,Bcl 2蛋白与尖锐湿疣的发病有密切关系 ,凋亡基因的表达可能抑制HPV的增殖     

Dendritic cell subsets and immunological milieu in inflammatory human papilloma virus-related skin lesions

Background

Human papilloma virus (HPV)-related warts persist, evading host immune surveillance, but sometimes disappear with inflammation.

Objectives

To elucidate the immune evasion mechanisms of HPV, we have examined the density, dynamics, and subsets of dendritic cell (DC) types in non-inflammatory or inflammatory HPV-related skin lesions such as warts and Bowen's disease (HPV-Bowen), and compared the epidermal expression levels of macrophage inflammatory protein (MIP)-3α and E-cadherin.

Methods

The expression of various DC markers, MIP-3α, and E-cadherin in the tissue samples obtained from patients with warts, HPV-Bowen and HPV-unrelated skin diseases was evaluated by immunohistochemistry. MIP-3α gene expression levels were examined in warts and HPV-Bowen by in situ hybridization (ISH) and real-time quantitative polymerase chain reaction (RT-qPCR).

Results

The numbers of Langerhans cells (LCs) and the expression levels of MIP-3α and E-cadherin were decreased in non-inflammatory warts and HPV-Bowen, as compared with normal skin. Both epidermal LCs and MIP-3α expression reappeared in inflammatory warts, associated with dermal infiltrates composed of many cytotoxic T cells and various subsets of DCs, while cellular infiltrates in HPV-Bowen contained many B cells and plasma cells with sparse infiltration of DCs. The upregulation of MIP-3α gene expression was confirmed in the inflammatory warts and HPV-Bowen by ISH and RT-qPCR.

Conclusions

The depletion of LCs in the non-inflammatory warts and HPV-Bowen is associated with a down-regulation of expression levels of MIP-3α and E-cadherin in the lesional keratinocytes. MIP-3α expression is upregulated in lesional keratinocytes of inflammatory warts, with the subsequent recruitment of various DC subsets and cytotoxic T cells, whereas plasma cell-rich infiltration was induced in HPV-Bowen.     

Determination of 8-methoxypsoralen levels in plasma and skin suction blister fluid by a new sensitive fluorodensitometric method

Summary An improved fluorodensitometric assay for the determination of 8-methoxypsoralen (8-MOP) in plasma is described. Because of its low limit of detection (below 1 ng/spot) this method is suitable to determine the drug in skin suction blister fluid, too. The standard deviation of the procedure is 6.4% or less.Plasma and skin blister fluid levels of 8-MOP are determined 2 h following oral administration of 40–60 mg 8-MOP. They range from 0–239 ng/ml and 0–163 ng/ml, respectively. A rather close correlation (r=0.91) between these two parameters could be observed.Thus, in cases with relatively high plasma levels sufficient skin levels can be predicted. If further investigations would prove, however, that a distinct concentration threshold required for therapeutic success exists — and recent experiments with fibroblast cultures imply that — skin blister fluid level determinations would seem highly desirable when plasma levels let us expect skin levels in the critical range.In general determination of 8-MOP skin blister fluid levels can be looked upon as a model for the evaluation of drug skin levels after systemic application in man.Supported in part by the Deutsche Forschungsgemeinschaft and by the Doktor-Robert-Pfleger-Stiftung     

Substance P restores normal skin architecture and reduces epidermal infiltration of sensory nerve fiber in TNCB-induced atopic dermatitis-like lesions in NC/Nga mice

Background

Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by intense pruritus and eczematous lesion. Substance P (SP) is an 11-amino-acid endogenous neuropeptide that belongs to the tachykinin family and several reports recently have supported the anti-inflammatory and tissue repairing roles of SP.