Evidence for local control of gene expression in the epidermal differentiation complex

The epidermal differentiation complex (EDC), located on chromosomal band 1q21, consists of at least 43 genes that are expressed during keratinocyte differentiation. Indicative of a role for chromatin structure in tissue specificity of EDC gene expression, we identified an inverse correlation between expression and DNA methylation for two EDC genes (S100A2 and S00A6) in human keratinocytes and fibroblasts. 5-azacytidine (5AC) and sodium butyrate (NaB) are two agents known to promote 'open' chromatin structure. To explore the relationship between chromatin structure and keratinocyte differentiation, we treated normal human keratinocytes (NHK) with 5AC or NaB, or with protocols known to promote their terminal differentiation. We then measured the steady-state mRNA levels for several S100 genes, small proline rich region-1, -2, and -3, loricrin, and involucrin by Northern blotting. 5AC and NaB each markedly increased expression of SPRR1/2 and involucrin in NHK. In contrast, expression of S100A2 was reduced by both agents, and by induction of keratinocyte differentiation. Moreover, while the clustered EDC genes displayed a general tendency to be expressed in epithelial cells, they displayed different patterns of cell type-specific expression. These results indicate that local, gene-specific factors play an important role in the regulation of EDC gene expression in the keratinocyte lineage and during keratinocyte terminal differentiation.     

Suppression of vitamin D receptor and induction of retinoid X receptor alpha expression during squamous differentiation of cultured keratinocytes

To gain more insight in the role of the vitamin D system in epidermal differentiation, we studied the expression of the vitamin D receptor and its heterodimeric partner retinoid X receptor alpha in cultured normal human keratinocytes during squamous differentiation, as triggered by different approaches. Northern and western blot analysis allowed us to investigate mRNA and protein levels of these nuclear receptors and of markers for growth control (c-myc, cyclin D1, p21WAF1) and differentiation (keratinocyte transglutaminase, small proline rich proteins). Growing cells to postconfluence was a potent stimulus for growth arrest and differentiation with concomitant suppression of vitamin D receptor and induction of retinoid X receptor alpha, at both the mRNA and the protein level. These changes could be prevented by concomitant treatment with epidermal growth factor or keratinocyte growth factor. Subjecting the cells to a calcium switch leading to stratification and differentiation lowered vitamin D receptor protein levels without affecting vitamin D receptor mRNA and induced both retinoid X receptor alpha mRNA and protein. Interferon-gamma and the phorbolester 12-O-tetradecanoyl phorbol 13-acetate, two well-known inducers of keratinocyte differentiation, both inhibited vitamin D receptor expression but only interferon-gamma induced retinoid X receptor alpha. The decreased vitamin D receptor expression was accompanied by reduced vitamin D responsiveness (as assessed by 24-hydroxylase mRNA induction) in postconfluent, high calcium, and 12-O-tetradecanoyl phorbol 13-acetate treated keratinocytes but not with interferon-gamma treatment. Taken together, our results associate vitamin D receptor expression with undifferentiated, proliferating keratinocytes, whereas retinoid X receptor alpha expression appears to be related to the differentiated phenotype. Therefore, proliferating and differentiating keratinocytes may be differentially targeted by active vitamin D metabolites.     

Identification of novel genes for secreted and membrane-anchored proteins in human keratinocytes

BACKGROUND: Both intercellular and intracellular signals are transduced primarily by interactions of secreted and/or membrane-anchored polypeptides, and they play a pivotal role in regulating proliferation, differentiation and apoptosis of keratinocytes within the epidermis. Despite recent identification of these polypeptides, it is likely that several important molecules remain undisclosed. OBJECTIVES: To identify novel genes encoding secreted or membrane-anchored polypeptides expressed by human keratinocytes. METHODS: We employed a signal sequence (SS) trap of a 5'-end-enriched cDNA library prepared from primary cultured human keratinocytes. Gene expression analysis was performed using Northern blotting. R Screening of 4018 cDNA clones yielded 82 positive clones (57 independent genes), most of which encoded SSs in their N-termini. Most of the positive clones were known genes registered in the GenBank database. Seven genes were identified in the EST database, four of which encoded novel membrane-anchored polypeptides with features of type I transmembrane proteins; the other three genes encoded novel non-type I transmembrane polypeptides. These EST genes were expressed differentially by keratinocytes subjected to low vs. high calcium concentrations and by basal vs. squamous cell carcinomas. CONCLUSIONS: Using the SS trap, we isolated many genes known to be involved in constituting epidermal structures and others that had not previously been associated with keratinocytes. In addition, we identified novel genes (EST genes) that differ in kinetics of gene expression in keratinocyte differentiation. Our results validate the effective use of this SS trap method for identifying secreted and membrane-anchored polypeptides expressed by human keratinocytes. The identification will better illuminate the molecular mechanisms responsible for co-ordinated regulation of epidermal homeostasis.     

nicastrin基因沉默的HaCaT细胞基因表达谱分析

【摘要】 目的 通过沉默人永生化角质形成细胞HaCaT细胞中nicastrin（NCSTN）基因的表达,研究其下游细胞增殖及分化相关信号通路的改变。方法 将HaCaT细胞分为干扰组、阴性对照组和空白对照组：干扰组转染特异性NCSTN-siRNA,阴性对照组转染阴性对照siRNA,空白对照组转染等量转染试剂。实时PCR及Western印迹检测各组NCSTN mRNA和蛋白表达验证转染效率。运用Agilent人类全基因组表达谱芯片技术研究干扰组HaCaT细胞基因表达谱与阴性对照组之间的差异,以表达上调或者下调倍数 ≥ 2.0且P ≤ 0.05为标准,将差异基因用GO分析进行富集,筛选出表达差异显著且与角质形成细胞增生分化相关的基因,用实时PCR验证结果。结果 干扰组HaCaT细胞NCSTN mRNA及蛋白相对表达量分别为0.287 ± 0.090、0.443 ± 0.085,明显低于阴性对照组（0.969 ± 0.127、1.047 ± 0.114）以及空白对照组（1.000 ± 0.151、1.000 ± 0.111）,差异均有统计学意义（F值分别为30.787、31.139,P值均为0.001）。表达谱芯片显示,与阴性对照组相比,干扰组表达下调基因605条,上调基因444条。GO分析显示,干扰后差异表达基因富集到上皮发育、上皮细胞分化、角质形成细胞分化、角化4种生物学过程。对表达差异显著且与角质形成细胞增生分化相关的Sprouty相关蛋白2基因、表皮生长因子7基因、胰岛素样生长因子结合蛋白5基因、人Rho关联含卷曲螺旋蛋白激酶2基因和骨形成发生蛋白6基因通过实时PCR验证,验证结果与芯片结果显示的差异趋势一致。结论 NCSTN基因功能缺失有可能通过调节其下游细胞增殖及分化相关信号通路的表达,影响角质形成细胞的正常增殖和分化。     

rhPTH(1-34)诱导的角质形成细胞Hedgehog信号通路差异表达基因的筛选

目的利用PCR芯片技术,研究重组人甲状旁腺素(1-34)[recombinanthumanparathyroidhormone,rhPTH(1-34)]对角质形成细胞Hedgehog(Hh)信号通路的影响,探讨其治疗银屑病的机制。方法培养HaCaT角质形成细胞株,用rhPTH(1-34)诱导细胞后,抽提rhPTH(1-34)组和空白对照组细胞的总RNA,用紫外吸收测定法和变性琼脂糖凝胶电泳法进行RNA产量和质量检测;合成cDNA,与含有84条人类基因的Hh信号通路的基因芯片进行杂交,并进行实时定量PCR扩增,最后对所得数据进行处理和分析。结果在84条基因中,rhPTH(1-34)组与空白对照组比较,25条基因表达差异2倍以上,其中23条显著上调,2条显著下调。这些差异表达基因涉及细胞增殖与分化、胚胎形成,细胞信号传导和多组织器官发育等。结论 rhPTH(1-34)可能通过影响Hh信号通路中PTCHD1,C6orf138,BMP4,BMP6,BMP7等相关基因表达,对角质形成细胞的增殖分化发挥调节作用。     

Expression of lysosome-associated membrane protein 1 (Lamp-1) and galectins in human keratinocytes is regulated by differentiation

Lysosomes and their components are suspected to be involved in epidermal differentiation. In this study, lysosomal enzyme activities, expression of the lysosome-associated membrane protein 1 (Lamp-1) and expression of the epidermal galectins-1, -3 and -7 were investigated in human keratinocytes cultured at different cell densities (subconfluence, confluence and postconfluence) in order to induce differentiation. Detected by Western blot and immunofluorescence, Lamp-1 expression is transiently upregulated at culture confluence, but reduced at postconfluence. Northern blot analyses performed on subconfluent, confluent and post-confluent cultures of keratinocytes show that Lamp-1 mRNA expression is also upregulated at culture confluence, but downregulated at postconfluence. Measurements of lysosomal enzyme activities indicate a transient upregulation at culture confluence, whereas cathepsins B, C and L are particularly downregulated at postconfluence. Cell density and differentiation of epidermal cells also differentially regulates galectin expression in autocrine cultures. As the expression of galectin-1 mRNA is high in subconfluent cells, it is assumed to be associated with their proliferative state. On the other hand, as the mRNA levels for galectins-3 and -7 are notably upregulated at culture confluence (galectin-7) or at postconfluence (galectin-3), their expression is thought to be related to the differentiated state of keratinocytes. However, we collected evidence by confocal microscopy that galectin-3 and Lamp-1 do not colocalize in vitro in keratinocytes. Altogether, our results suggest that the upregulated Lamp-1 expression at confluence could be involved in keratinocyte differentiation, but apparently not through interaction with galectin-3.     

Gene expression profiles of three different models of reconstructed human epidermis and classical cultures of keratinocytes using cDNA arrays

The gene expression profiles of three different models of reconstructed human epidermis were analyzed in a comparative study using cDNA array technology. The study also included normal human subconfluent keratinocytes cultured on plastic. Arrays were custom-made and comprised 504 known genes related to cutaneous biology. The gene expression profiles of the three reconstructed epidermis models shared 86% similarity; only 22 of the 504 examined genes showed a different expression level. A comparison of the 3D models with keratinocyte cultures on plastic dishes revealed a set of six genes with a considerably higher expression in the 3D models. These genes were keratin 1, corneodesmosin, filaggrin, loricrin, calmodulin-like skin protein and caspase 14, all related to keratinocyte terminal differentiation. The reported data may contribute to a better understanding and characterization of reconstructed epidermal models and may also serve as established references for investigations related to epidermal differentiation and proliferation.     

Stratum basale keratinocyte expression of the cell‐surface glycoprotein CDCP1 during epidermogenesis and its role in keratinocyte migration

Background Epidermogenesis and epidermal wound healing are tightly regulated processes during which keratinocytes must migrate, proliferate and differentiate. Cell‐to‐cell adhesion is crucial to the initiation and regulation of these processes. CUB‐domain‐containing protein (CDCP)1 is a transmembrane glycoprotein that is differentially tyrosine phosphorylated during changes in cell adhesion and survival signalling, and is expressed by keratinocytes in native human skin, as well as in primary cultures. Objectives To investigate the expression of CDCP1 during epidermogenesis and its role in keratinocyte migration. Methods We examined both human skin tissue and an in vitro three‐dimensional human skin equivalent model to examine the expression of CDCP1 during epidermogenesis. To examine the role of CDCP1 in keratinocyte migration we used a function‐blocking anti‐CDCP1 antibody and a real‐time Transwell? cell migration assay. Results Immunohistochemical analysis indicated that in native human skin CDCP1 is expressed in the stratum basale and stratum spinosum. In contrast, during epidermogenesis in a three‐dimensional human skin equivalent model, CDCP1 was expressed only in the stratum basale, with localization restricted to the cell–cell membrane. No expression was detected in basal keratinocytes that were in contact with the basement membrane. Furthermore, an anti‐CDCP1 function‐blocking antibody was shown to disrupt keratinocyte chemotactic migration in vitro. Conclusions These findings delineate the expression of CDCP1 in human epidermal keratinocytes during epidermogenesis and demonstrate that CDCP1 is involved in keratinocyte migration.     

Pro‐differentiating effects of oxysterols in keratinocytes

Oxysterols stimulate keratinocyte differentiation, which involves the formation of the cornified envelope on the inner plasma membrane by transglutaminase crosslinking of several constituent proteins. Oxysterols increase the expression of one of these crosslinked proteins, involucrin, which can be abolished by mutations of the distal activator protein (AP)‐1 response element in the involucrin promoter. Here, we show that oxysterols increase AP‐1 binding in electrophoretic gel mobility shift assays and that oxysterols induce the expression of an AP‐1 reporter. We further describe the individual components of the AP‐1 complex, which are involved in the oxysterol‐mediated AP‐1 activation and stimulation of keratinocyte differentiation. We identified Fra‐1 within the AP‐1 DNA binding complex by super shift analysis of nuclear extracts from oxysterol‐treated, cultured keratinocytes. Western blot analysis demonstrated that oxysterol treatment increased the levels of Fra‐1 and Jun‐D, while Northern analysis revealed that oxysterols increased mRNA levels for Fra‐1, c‐Fos and Jun‐D. Together these data indicate that oxysterols stimulate involucrin expression by increasing the levels of specific proteins of the AP‐1 complex, indicating that oxysterols regulate keratinocyte differentiation by inducing the AP‐1 factors, which in turn activate the genes required for epidermal differentiation. The presence of LXREs within the promoter regions of individual AP‐1 proteins suggests that these oxysterol effects may be mediated by LXR.     

HAX-1, identified by differential display reverse transcription polymerase chain reaction,is overexpressed in lesional psoriasis

Psoriasis is a chronic inflammatory disease characterized by epidermal hyperplasia and an inflammatory infiltrate. The normal differentiation from basal to granular keratinocytes with subsequent apoptosis and cornification is disturbed in the akanthotic epidermis. This could be due to both an excess of mitogenic stimuli with hyperproliferation and/or resistance to apoptosis. By mRNA differential display we found HAX-1 to be overexpressed in lesional psoriatic skin. The overexpression of HAX-1 was verified at the mRNA level by Northern blot and in situ hybridization, as well as at the protein level by Western blot and immunohistochemistry. Detection of HAX-1 in mRNA from different tissues showed strong expression in the brain, pancreas, skeletal muscle, and heart. In contrast to primary keratinocytes and melanocytes we found HAX-1 highly expressed in human immortalized keratinocytes (HaCaT) and different melanoma cell lines. In HaCaT cells as a model for psoriatic keratinocytes we found an increased ultraviolet-induced apoptosis after expression of HAX-1 antisense mRNA. In psoriasis, the epidermal differentiation could be disturbed due to the increased expression of HAX-1 and hence a prolonged resistance to terminal differentiation. Antiapoptotic mechanisms are an emerging concept for the understanding of the pathogenesis of this disease possibly leading to clinical applications.     

Molecular cloning and expression of human keratinocyte proline-rich protein (hKPRP), an epidermal marker isolated from calcium-induced differentiating keratinocytes

We isolated a human gene encoding keratinocyte proline-rich protein (hKPRP). hKPRP gene is located in the region of epidermal differentiation complex on chromosome 1q21, and its approximately 2.5 kb mRNA encodes 579 amino acid protein with high proline content (18%). The mRNA level of hKPRP was markedly increased at both 7 and 14 d after treatment with 1.2 mM calcium in cultured normal human epidermal keratinocytes. In situ hybridization demonstrated that hKPRP was expressed in upper granular layer of normal epidermis with characteristic intermittent pattern. In psoriatic lesion, hKPRP expression was increased as compared with normal skin and showed continuous pattern. Immunohistochemical analysis also confirmed the expression of hKPRP at the protein level. Western blot analysis showed that hKPRP protein of approximately 70 kDa size was significantly increased by calcium in a time-dependent manner. In mouse tissue blot assays, the expression of KPRP was detected in stomach and skin tissues, and began at 17.5 embryonic days. Additionally, hKPRP expression was detected in the periderm of human fetal skin from 16 wk estimated gestational age. Together, these results suggest that hKPRP is an epidermal marker expressed in stratified squamous epithelia and has a potential role in keratinocytes differentiation.     

Plasminogen activator inhibitor type 2 is expressed in keratinocytes during re-epithelialization of epidermal defects

Plasminogen activation is observed in the human epidermis during re-epithelialization of epidermal defects. The activation reaction depends on plasminogen activators (PAs) associated with re-epithelializing keratinocytes. PA inhibitor type 2 (PAI-2) is thought to be a major epidermal PA inhibitor in keratinocytes. However, no data are available on the expression of PAI-2 in keratinocytes during epidermal regeneration. We have therefore analysed PAI-2 at the mRNA and protein level in keratinocyte cultures as well as in epidermal lesions in which re-epithelializing keratinocytes were apparent. We found that PAI-2 expression at the mRNA and protein level was negatively correlated with the cell density in regular keratinocyte cultures. In organotypic cocultures, in which the transition from a re-epithelializing to a sedentary phenotype can be studied, PAI-2 was most strongly expressed in early cultures prior to formation of a differentiated epidermis-like structure. We found a strong expression of PAI-2 in keratinocytes that re-epithelialized dermal burn wounds or lesions caused by the autoimmune blistering disease pemphigus vulgaris. Our results suggest that not only PAs, but also a major PA inhibitor, PAI-2, are expressed in keratinocytes that are actively involved in re-epithelialization.