BNIP3 promotes the motility and migration of keratinocyte under hypoxia

The migration of keratinocytes from wound margins plays a critical role in the re‐epithelialization of skin wounds. Hypoxia occurs immediately after injury and acts as an early stimulus to initiate the healing processes. Although our previous studies have revealed that hypoxia promotes keratinocyte migration, the precise mechanisms involved remain unclear. Here, we found that BNIP3 expression was upregulated in hypoxic keratinocytes, and BNIP3 silencing suppressed hypoxia‐induced cell migration. Additionally, hypoxia activated the focal adhesion kinase (FAK) pathway through upregulation of BNIP3, while FAK inhibition attenuated hypoxic keratinocyte migration. Here, we conclusively demonstrate a novel role for BNIP3 in hypoxia‐induced keratinocyte migration. Furthermore, we provide a new perspective on the molecular mechanisms of wound healing and identify BNIP3 as a potential new molecular target for clinical treatments to enhance wound healing.     

角质形成细胞旁分泌作用在瘢痕疙瘩形成中的研究进展

角质形成细胞是表皮的主要组成细胞,其可分泌多种细胞因子,对临近细胞产生生物学影响。角质形成细胞旁分泌在瘢痕疙瘩的形成中发挥了一定作用,本文从角质形成细胞旁分泌在瘢痕疙瘩生物学发病机制中的作用做一综述。     

Signals that initiate, augment, and provide directionality for human keratinocyte motility

Human keratinocytes (HK) migration plays a critical role in the re-epithelialization of acute skin wounds. Although extracellular matrices (ECM) and growth factors (GF) are the two major pro-motility signals, their functional relationship remains unclear. We investigated how ECM and GF regulate HK motility under defined conditions: (1) in the absence of GF and ECM and (2) with or without GF with cells apposed to a known pro-motility ECM. Our results show that HK migrate on selected ECM even in the total absence of GF. This suggests that certain ECM alone are able to "initiate" HK migration. Unlike ECM, however, GF alone cannot initiate HK migration. HK cannot properly migrate when plated in the presence of GF, regardless of the concentration, without an ECM substratum. The role of GF, instead, is to augment ECM-initiated motility and provide directionality. To gain insights into the mechanism of action by ECM and GF, we compared, side-by-side, the roles of three major mitogen-activated protein kinase cascades, extracellular-signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK). Our data show that ERK1/2 is involved in mediating collagen's initiation signal and GF's augmentation signal. p38 is specific for GF's augmentation signal. JNK is uninvolved in HK motility. Constitutively activated p38 and ERK1/2 alone could not initiate HK migration. Co-expression of both constitutively activated p38 and ERK1/2, however, could partially mimic the pro-motility effects of collagen and GF. This study reveals for the first time the specific functions of ECM and GF in cell motility.     

姜黄油对角质形成细胞体外增殖分化影响的研究

目的：探讨姜黄油对角质形成细胞体外增殖分化的影响。方法：以人角质形成细胞株COLO-16细胞为模型，噻唑盐比色法(MTT)检测细胞活性和生长情况；免疫组化SABC法检测姜黄油对增殖细胞核抗原(PCNA)表达的影响；电镜观察姜黄油对细胞超微结构的影响。结果：姜黄油抑制角质形成细胞增殖，随药物浓度增加，其抑制增殖能力增加，PCNA表达逐渐减弱，且对角质形成细胞超微结构有直接损伤作用。结论：姜黄油具有抑制体外角质形成细胞增殖分化的作用，有望成为一种治疗增殖性皮肤病的外用药物。     

紫草素对角质形成细胞株增殖的抑制及凋亡的作用

目的：探讨紫草素对角质形成细胞株colo-16增殖及凋亡的调节作用。方法：利用体外细胞培养技术、MTT法、流式细胞仪、荧光显微镜技术检测不同浓度的紫草素对colo-16细胞株增殖及凋亡的影响。结果：紫草素对colo-16细胞株的作用与浓度及时间呈一定的关系，浓度越高，时间越长，其抑制作用越强。终浓度为0．25μg/ml、0．5μg/ml的紫草素，培养48h后，细胞的凋亡率分别为9．89％、25．04％，正常对照组为5．86％，两者相比差异显著。结论：紫草素值得进一步深入地研究与探讨。     

Immunomodulatory effects of a set of amygdalin analogues on human keratinocyte cells

Peptide T (PT) is an octapeptide shown to resolve psoriatic lesions. Our previous investigations suggest that keratinocytes play an important role in conditioning the therapeutic effects of the PT in psoriasis. However, peptides are not good therapeutic agents, because they exhibit poor absorption, are easily metabolized and are immunogenic. Using computational methods, the natural product amygdalin was identified as peptidomimetic of PT. However, amygdalin exhibits a toxic profile due to its cyanide group. To overcome this deleterious effect, we synthesized analogues lacking the cyanide group. Human keratinocytes were treated with PT or with three different peptidomimetics of PT. To study its effects on the expression of HSP-70, TGF-beta, alpha-v integrin, ICAM-1 and cytokines, we analysed the protein levels by Western blot and ELISA. Our results show that the different peptidomimetics of PT tested exhibit a similar biological behaviour in regard to the overexpression of HSP-70, TGF-beta and alpha-v integrin than the native peptide. TNF-alpha is overexpressed by PT and SVT-03018; between the other two analogs, SVT-03016 do not produce any significant change in regard to the control, while SVT-03017 shows only a moderate increase in regard to control. SVT-03018 provokes a remarkable upregulation of IL-10, stronger than SVT-03016, SVT-03017 and PT. All the other three analogues reduce comparably to the PT, the expression of ICAM-1 and do not increase the release of proinflammatory cytokines. The results highlighted that the three analogues of amygdalin with the cyanide group removed exhibit the same biological effects of PT. Therefore, they can be considered peptidomimetics, suggesting their possible use in the treatment of psoriasis.     

角质形成细胞生长因子对HaCaT细胞增殖的影响

目的:研究角质形成细胞生长因子(KGF)对HaCaT细胞增殖的影响,探讨KGF与银屑病表皮过度增殖的关系。方法:利用HaCaT细胞体外培养,采用KGF为增殖诱导剂,四甲基偶氮唑盐比色法(MTT)检测不同浓度的KGF对HaCaT细胞增殖率的影响;检测KGF对细胞生长曲线及细胞集落形成的影响。结果:MTT实验表明,KGF可刺激HaCaT细胞增殖,刺激作用呈浓度依赖性(r=0.981,P<0.05);10 ng/mL KGF作用下HaCaT细胞的生长速度较对照组明显加快(P<0.05),集落形成率较对照组增加(P<0.05),且细胞呈明显的增殖形态,形成的集落较大,集落内细胞多呈星状,胞内染色质丰富。结论:KGF具有显著的促HaCaT细胞增殖作用,银屑病皮损区域KGF表达增加可能是引起表皮过度增殖的重要致病因子。     

重组人IL－6对角朊细胞体外增殖分化影响的研究

为寻找一种对角朊细胞增殖分化具有调节作用的细胞因子，以人转化型角朊细胞株Ｃｏ－ｌｏ－１６细胞为模型，通过活细胞计数，３Ｈ－ＴｄＲ掺入法，细胞因子生物活性检测、电镜及免疫组化方法研究了重组人白细胞介素－６对角朊细胞体外增殖分化的影响。结果表明，ＩＬ－６对角朊细胞的体外增殖和ＤＮＡ合成具有双向调节作用；大剂量的ＩＬ－６能减少角朊细胞肿瘤坏死因子－α、ＩＬ－６的产生；对角朊细胞胞膜、线粒体有直接损伤作用；并抑制角朊细胞角蛋白表达。提示ＩＬ－６可作为角朊细胞体外增殖分化的免疫调节因子。     

Crucial effects of fibroblasts and keratinocyte growth factor on morphogenesis of reconstituted human oral epithelium

The connective tissue is known to have a general supportive effect for the development of the overlying epithelium; however, the more specific effects of fibroblasts and the involvement of their product, keratinocyte growth factor, on oral epithelial morphogenesis have not yet been addressed. Therefore, the purpose of this study was to investigate the effects of fibroblasts and keratinocyte growth factor on human oral epithelial morphogenesis in vitro. Reconstituted human oral epithelium was generated from primary human oral keratinocytes and fibroblasts by use of an organotypic cell culture model in a defined medium. Addition of fibroblasts to the collagen biomatrix increased total epithelial thickness from 28.0+/-5.0 microm to 66.1+/-8.6 microm (p=0.028), and basal cell proliferation from 3.6+/-0.7% to 16.6+/-1.1% (p=0.025). Presence of fibroblasts profoundly influenced the pattern of epithelial differentiation, and induced a switch in the pattern of cell death, from a predominance of spontaneous cell death in the basal cell layer (from 4.7+/-0.6% to 1.8+/-0.3%, p=0.029) to a more prevalent cell death due to terminal differentiation in the suprabasal cell layer (from 4.0+/- 0.1% to 5.4+/-0.1%, p=0.034). Keratinocyte growth factor promoted epithelial growth, but did not significantly enhance epithelial differentiation, demonstrating that fibroblasts possess additional mechanisms to keratinocyte growth factor synthesis that can modulate differentiation of reconstituted human oral epithelium.     

Reversible inhibition of keratinocyte thymidine incorporation by the calmodulin antagonist, W-7

Although calmodulin has been suggested as an important regulator of keratinocyte proliferation, its precise role remains unknown. We employed a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), to examine the role of calmodulin on keratinocyte proliferation. N-(6 aminohexyl-1-naphthalenesulfonamide (W-5), a chlorine-deficient analogue of W-7 with little anti-calmodulin activity, was used as the control. W-7 markedly inhibited thymidine incorporation of pig epidermis at concentrations close to its anti-calmodulin activity; W-5 had no effect on the thymidine incorporation. The inhibitory effect of W-7 was reversible; the removal of W-7 from the incubation medium resulted in the reinitiation of the thymidine incorporation, suggesting that W-7 is not a cytotoxic agent. These results are consistent with the view that calmodulin is an essential regulator of keratinocyte proliferation. The epidermal beta-adrenergic response, which is decreased in various hyperproliferative epidermal abnormalities, was increased in W-7-treated hypoproliferative epidermis. The epidermal SOD activity, which is also decreased in the hyperproliferative epidermis, however, was not affected by the W-7 treatment.     

Human keratinocyte locomotion: the effect of selected cytokines.

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) are two powerful mitogens for human keratinocytes that also have been shown to promote the healing of in vivo wounds. Transforming growth factor-beta (TGF-beta) markedly inhibits human keratinocyte proliferation and growth and yet has been shown to promote wound healing. Using a migration assay that evaluates pure cell locomotion independently from cell proliferation, we examined the influence of EGF, bFGF, and TGF-B on human keratinocyte locomotion. Although these agents had profound influences upon the growth potential of keratinocytes in parallel thymidine incorporation assays, they had no significant effect upon keratinocyte locomotion when cells were apposed to either tissue culture plastic or a collagen substratum. In contrast, we found that bovine pituitary extract (BPE), a poorly defined mitogen that is commonly used in keratinocyte cultures, could stimulate keratinocyte locomotion when the cells were apposed to a collagen substrate. These studies demonstrate that i) keratinocyte locomotion and proliferation operate by completely independent mechanisms, ii) the positive effects upon wound healing by EGF, bFGF, and TGF-beta are not due to a direct promotion of keratinocyte locomotion, and iii) that one or more components of BPE are capable of directly promoting keratinocyte locomotion on collagen.     

异氟烷对人精子运动功能和顶体反应影响研究

目的:观察异氟烷对人精子运动功能、体外获能以及精子顶体反应的影响,对有生育要求的男性提供临床用药依据。方法:收集我院男科门诊就诊的男性精子标本,从中筛选出18份活力正常精子的精液作为标本,每例精液均分成3份,分别放置于3个试管中,分别给予1.1%异氟烷(ISO1组)、2.2%异氟烷(ISO2组)和空气作为对照组,把所有的有机玻璃箱放置于孵箱培养5h,对精子的运动、获能、顶体反应进行评价。结果:1.1%和2.2%浓度的异氟烷作用于精子5h以后,与对照组相比,精子的运动活力、直线速度、曲线速度、平均速度均明显下降,P<0.05,差异有统计学意义,ISO1组和ISO2组比较,ISO2组的下降更为明显,呈现剂量依赖关系,即随着浓度的升高,精子的运动功能抑制越明显。对于精子获能的影响,与对照组比较,两种浓度的异氟烷均明显地抑制精子的体外获能,P均<0.05,差异均有统计学意义,ISO1组和ISO2组比较无明显差异,P>0.05。两组精子发生顶体反应的百分率均较处理前明显增加,P<0.05,差异有统计学意义。但是处理后两组间比较无明显差异。结论:异氟烷能够抑制精子的运动和获能,并且随着浓度的增加,抑制作用增强;异氟烷对精子顶体反应影响不明显。     

Differential effects of retinoids on DNA synthesis in calcium-regulated murine epidermal keratinocyte cultures

To study the possibility that the state of proliferation of epidermal keratinocytes can influence the action of retinoids, the rate of proliferation of murine epidermal keratinocytes was manipulated by growing the cells in media containing high or low concentrations of Ca++. In contrast to what other investigators have reported, keratinocytes cultured in medium containing 1.4 mM Ca++ proliferate faster, instead of slower, than cells cultured in medium with 0.09 mM Ca++. Other experiments showed that Ca++ was stimulatory to keratinocytes in medium containing a low level of growth factors, and inhibitory in medium containing a high level of growth factors, suggesting that the discrepancy could be due to a difference in the sera used. The high Ca++ cells prominently expressed the 48kD/56kD pair of keratin, showing that they were in a hyperproliferative state. Exposure of the faster growing high Ca++ cells to all-trans retinoic acid, 13-cis retinoic acid, etretinate, etretin, and arotinoid ethyl ester caused dose-dependent inhibition of DNA synthesis. In contrast, exposure of the slower growing low Ca++ cells to these retinoids resulted in dose-dependent stimulation of DNA synthesis. In addition, all-trans retinoic acid caused dose-related increases in cell number in the low Ca++ cultures. These findings correlate with the reported differential effects of retinoids on normal and hyperproliferative epidermis, and suggest that Ca++ and low growth factor-regulated keratinocyte cultures are useful for studying the mechanism of hyperproliferation and retinoid actions.     

Adenosine- and adenine-nucleotide-mediated inhibition of normal and transformed keratinocyte proliferation is dependent upon dipyridamole-sensitive adenosine transport

Extracellular adenosine and its related nucleotides have been referred to as retaliatory metabolites that can be released into the extracellular environment during inflammation, wounding, and other pathologic states. We have previously reported that these compounds reversibly inhibit the proliferation of normal keratinocyte cultures and we now demonstrate that these compounds also arrest the proliferation of transformed keratinocytes. Although our study shows that keratinocytes express mRNA corresponding to the A2B purinoreceptors and that adenosine or AMP treatment elevates intracellular cAMP in these cells, our study also demonstrates that dipyridamole-inhibitable transport of adenosine into the keratinocyte is central to the mechanism by which adenosine and adenine nucleotides arrest proliferation in these cells. In support of this mechanism, our results demonstrate that human keratinocytes express mRNA corresponding to the recently cloned dipyridamole-sensitive human equilibrative nucleoside transporter. Interestingly, coincubation with adenosine deaminase reverses the antiproliferative action of adenosine and exerts no effect on the antiproliferative activity of the adenine nucleotides, thus supporting a model in which adenine nucleotides are enzymatically converted to adenosine and transported into the keratinocyte in a tightly coupled and adenosine-deaminase-resistant manner. Analysis of adenosine- and adenosine-monophosphate-treated keratinocytes demonstrated that quiescence is induced within 12-24 h, and fluorescence-activated cell sorter analysis suggests that treatment with these compounds may result in the inhibition of keratinocyte proliferation at both G1 and S phases of the cell cycle. In addition to their documented antiproliferative action on other cell types, adenosine, adenine nucleotides, and related analogs may also represent a potential new class of pharmacologic regulators of keratinocyte proliferation in vivo.