H-Ras Expression in Immortalized Keratinocytes Produces an Invasive Epithelium in Cultured Skin Equivalents

Background

Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent.

Methodology/Principal Findings

Previously described cdk4 and hTERT immortalized foreskin keratinocytes were engineered to express ectopically introduced H-ras. Skin equivalents, composed of normal fibroblast-contracted collagen gels overlaid with keratinocytes (immortal or immortal expressing H-ras), were prepared and incubated for 3 weeks. Harvested tissues were processed and sectioned for histology and antibody staining. Antigens specific to differentiation (involucrin, keratin-14, p63), basement-membrane formation (collagen IV, laminin-5), and epithelial to mesenchymal transition (EMT; e-cadherin, vimentin) were studied. Results showed that H-ras keratinocytes produced an invasive, disorganized epithelium most apparent in the lower strata while immortalized keratinocytes fully stratified without invasive properties. The superficial strata retained morphologically normal characteristics. Vimentin and p63 co-localization increased with H-ras overexpression, similar to basal wound-healing keratinocytes. In contrast, the cdk4 and hTERT immortalized keratinocytes differentiated similarly to normal unimmortalized keratinocytes.

Conclusions/Significance

The use of isogenic derivatives of stable immortalized keratinocytes with specified genetic alterations may be helpful in developing more robust in vitro models of cancer progression.     

Testosterone Stimulates Duox1 Activity through GPRC6A in Skin Keratinocytes

Testosterone is an endocrine hormone with functions in reproductive organs, anabolic events, and skin homeostasis. We report here that GPRC6A serves as a sensor and mediator of the rapid action of testosterone in epidermal keratinocytes. The silencing of GPRC6A inhibited testosterone-induced intracellular calcium ([Ca²⁺]_i) mobilization and H₂O₂ generation. These results indicated that a testosterone-GPRC6A complex is required for activation of G_q protein, IP₃ generation, and [Ca²⁺]_i mobilization, leading to Duox1 activation. H₂O₂ generation by testosterone stimulated the apoptosis of keratinocytes through the activation of caspase-3. The application of testosterone into three-dimensional skin equivalents increased the apoptosis of keratinocytes between the granular and stratified corneum layers. These results support an understanding of the molecular mechanism of testosterone-dependent apoptosis in which testosterone stimulates H₂O₂ generation through the activation of Duox1.     

Pinx1表达与端粒酶活性及肿瘤的关系

Pinx1作为端粒酶抑制剂,也是最近发现的一种新型肿瘤抑制因子.通过对内源性端粒酶抑制基因Pinx1与端粒酶、端粒相关蛋白在肿瘤中的表达以及Pinx1在肿瘤演进过程中的作用及临床意义进行一系列的研究,发现肿瘤中Pinx1表达下降与端粒酶活性增高密切相关,且端粒酶活性增高的程度与肿瘤的预后相关.现对近年来Pinx1基因的最新研究进展,特别是该基因表达与端粒酶活性在肿瘤发生、发展中的作用,作一综述和分析.     

Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes

Purpose

Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed.

Materials and Methods

Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR.

Results

Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C.

Conclusion

HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture.     

Protective Effect of Indole-3-Pyruvate against Ultraviolet B-Induced Damage to Cultured HaCaT Keratinocytes and the Skin of Hairless Mice

Previous investigations demonstrated that pyruvate protects human keratinocytes against cell damage stemming from exposure to ultraviolet B (UVB) radiation. This study endeavoured to elucidate the protective capacity of aromatic pyruvates (e.g., phenylpyruvate (PPyr), 4-hydroxyphenylpyruvate (HPPyr), and indole-3-pyruvate (IPyr)) against UVB-induced injury to skin cells, both in vitro and in vivo. Cultured human HaCaT keratinocytes were irradiated with UVB light (60 mJ/cm²) and maintained with or without test compounds (1–25 mM). In addition, the dorsal skin of hairless mice (HR-1) was treated with test compounds (100 µmol) and exposed to UVB light (1 J/cm²) for two times. The ability of the test compounds to ameliorate UVB-induced cytotoxicity and inflammation was then assessed. Aromatic pyruvates reduced cytotoxicity in UVB-irradiated HaCaT keratinocytes, and also diminished the expression of interleukin 1β (IL-1β) and interleukin 6 (IL-6). IPyr was more efficacious than either PPyr or HPPyr. Furthermore, only IPyr inhibited cyclooxygenase-2 (Cox-2) expression at both the mRNA and the protein level in UVB-treated keratinocytes. Topical application of IPyr to the dorsal skin of hairless mice reduced the severity of UVB-induced skin lesions, the augmentation of dermal thickness, and transepithelial water loss. Overproduction of IL-1β and IL-6 in response to UVB radiation was also suppressed in vivo by the topical administration of IPyr. These data strongly suggest that IPyr might find utility as a UVB-blocking reagent in therapeutic strategies to lessen UVB-induced inflammatory skin damage.     

紫外线A照射差异性损伤皮肤角质形成细胞和成纤维细胞

紫外线A(ultraviolet A,UVA)照射皮肤可产生红斑、色素沉着以及光敏性损伤,导致光老化甚至诱导皮肤癌。研究发现,UVA(320～400 nm)照射对皮肤角质形成细胞和成纤维细胞造成损伤的反应和损伤程度都有所不同。本文结合近年来的相关研究,对UVA照射导致的上述两种细胞的形态、细胞膜损伤、血红素氧合酶(heme oxygenase,HO)活性、HO-1表达、细胞内有害自由铁形成,以及其它抗氧化物水平的不同反应等进行阐述,总结分析了同等剂量UVA照射后对角质形成细胞和成纤维细胞损伤的差异性,这些分析可能为皮肤抗UVA照射损伤以及寻找新型防晒剂的开发提供新思路和实验依据。     

Sphingomyelinase D from Loxosceles laeta Venom Induces the Expression of MMP7 in Human Keratinocytes: Contribution to Dermonecrosis

Envenomation by Loxosceles spider is characterized by the development of dermonecrosis. In previous studies, we have demonstrated that increased expression/secretion of matrix metalloproteinases 2 and 9, induced by Loxosceles intermedia venom Class 2 SMases D (the main toxin in the spider venom), contribute to the development of cutaneous loxoscelism. In the present study we show that the more potent venom containing the Class 1 SMase D from Loxosceles laeta, in addition to increasing the expression/secretion of MMP2 and MMP9, also stimulates the expression of MMP7 (Matrilysin-1), which was associated with keratinocyte cell death. Tetracycline, a matrix metalloproteinase inhibitor, prevented cell death and reduced MMPs expression. Considering that L. laeta venom is more potent at inducing dermonecrosis than L. intermedia venom, our results suggest that MMP7 may play an important role in the severity of dermonecrosis induced by L. laeta spider venom SMase D. In addition, the inhibition of MMPs by e.g. tetracyclines may be considered for the treatment of the cutaneous loxoscelism.     

Analysis of Telomerase Expression and Proliferative Activity in the Different Layers of Cyclic Endometrium

To gain better insights into cell kinetics under physiological conditions, telomerase activity in the functional and basal layers of cyclic endometrium (n= 33) was compared with the immunostaining of glandular and stromal cells within these layers (n= 25). Two immunohistochemical proliferation markers were used to demarcate cells in the G₁phase of the cell cycle. In contrast to previous expectations, telomerase activity and both glandular and stromal proliferative activities were all significantly higher in the functional than in the basal endometrium (P< 0.002). The course of telomerase activity in the endometrial layers during the ovarian cycle was significantly associated with the proliferative scores for the functional and basal endometrial glands and the functional stroma but not the stromal compartment of the basal layer. Our findings indicate that the telomerase activity in cyclic endometrium is associated with the total number of proliferating glandular and stromal cells in the functional layer. Proliferating daughter cells of telomerase-competent stem cells may account for the lower levels of telomerase detected in normal basal endometrium.     

FRAP Analysis Reveals Stabilization of Adhesion Structures in the Epidermis Compared to Cultured Keratinocytes

Proper development and tissue maintenance requires cell-cell adhesion structures, which serve diverse and crucial roles in tissue morphogenesis. Epithelial tissues have three main types of cell-cell junctions: tight junctions, which play a major role in barrier formation, and adherens junctions and desmosomes, which provide mechanical stability and organize the underlying cytoskeleton. Our current understanding of adhesion function is hindered by a lack of tools and methods to image junctions in mammals. To better understand the dynamics of adhesion in tissues we have created a knock-in ZO-1-GFP mouse and a BAC-transgenic mouse expressing desmoplakin I-GFP. We performed fluorescence recovery after photobleaching (FRAP) experiments to quantify the turnover rates of the tight junction protein ZO-1, the adherens junction protein E-cadherin, and the desmosomal protein desmoplakin in the epidermis. Proteins at each type of junction are remarkably stable in the epidermis, in contrast to the high observed mobility of E-cadherin and ZO-1 at adherens junctions and tight junctions, respectively, in cultured cells. Our data demonstrate that there are additional mechanisms for stabilizing junctions in tissues that are not modeled by cell culture.     

人类癌组织中端粒酶活性与p16基因表达的相关性

端粒酶是一种由RNA和蛋白质组成的RNA酶,依其自身序列逆转录合成端粒序列并添加到染色体末端,以保证细胞分裂的稳定性。近年来的研究已证实端粒酶的激活是许多恶性肿瘤发生的先决条件。p16基因作为一种重要的抑癌基因,其编码产物P16蛋白可通过p16-cyclinD/CDK-pRB途径调控细胞周期。在多种肿瘤中均出现p16基因表达异常,其表达缺失与人类肿瘤的发生发展关系密切。就端粒酶活性与p16基因表达在人类癌组织中的相关性及其可能作用机制进行总结分析。     

GSRd 对人脑胶质瘤U251 细胞端粒酶活性和hTERT表达的影响

目的:探索人参苷皂Rd对人脑胶质瘤的作用。方法:人脑胶质瘤U251细胞系细胞培养,不同浓度的人参皂苷Rd处理观察细胞形态、测定端粒酶活性以及h TERT表达水平。结果:随着GSRd浓度的升高,U251细胞的生长被明显抑制,出现了细胞凋亡和凋亡小体的现象;在经GSRd处理后U251细胞的端粒酶活性,对照组和溶媒组类似,而GSRd药物处理24 h后,细胞端粒酶活性显著降低,其中20μg/L组端粒酶活性降低极显著,P0.01,40μg/L和80μg/L组端粒酶活性显著降低,P0.05。GSRd药物处理48 h后,细胞端粒酶在20、40、80μg/L处理后,端粒酶活性降低极显著,P0.01;20μg/L、40μg/L和80μg/L的GSRd处理细胞后,相比于对照和溶媒组,h TERT基因表达水平显著降低。结论:人参皂苷Rd能够促进人脑胶质瘤U251细胞凋亡,对于临床治疗脑胶质瘤有重要的意义。     

POT1a and Components of CST Engage Telomerase and Regulate Its Activity in Arabidopsis

Protection of Telomeres 1 (POT1) is a conserved nucleic acid binding protein implicated in both telomere replication and chromosome end protection. We previously showed that Arabidopsis thaliana POT1a associates with the TER1 telomerase RNP, and is required for telomere length maintenance in vivo. Here we further dissect the function of POT1a and explore its interplay with the CST (CTC1/STN1/TEN1) telomere complex. Analysis of pot1a null mutants revealed that POT1a is not required for telomerase recruitment to telomeres, but is required for telomerase to maintain telomere tracts. We show that POT1a stimulates the synthesis of long telomere repeat arrays by telomerase, likely by enhancing repeat addition processivity. We demonstrate that POT1a binds STN1 and CTC1 in vitro, and further STN1 and CTC1, like POT1a, associate with enzymatically active telomerase in vivo. Unexpectedly, the in vitro interaction of STN1 with TEN1 and POT1a was mutually exclusive, indicating that POT1a and TEN1 may compete for the same binding site on STN1 in vivo. Finally, unlike CTC1 and STN1, TEN1 was not associated with active telomerase in vivo, consistent with our previous data showing that TEN1 negatively regulates telomerase enzyme activity. Altogether, our data support a two-state model in which POT1a promotes an extendable telomere state via contacts with the telomerase RNP as well as STN1 and CTC1, while TEN1 opposes these functions.