蛋白激酶D1及其磷酸化位点在皮肤鳞状细胞癌、Bowen病及光线性角化病组织中的表达

目的 探讨蛋白激酶DI(PKD1)及其磷酸化位点pPKD1-tyr463和pPKD1-ser916在鳞状细胞癌(SCC)、Bowen病和光线性角化病(AK)中的表达及意义.方法 收集新鲜SCC、Bowen病、AK及正常皮肤组织各10份,RT-PCR法检测各组样本中PKD1在基因水平的表达,Western印迹法检测各组样本中PKD1及其磷酸化位点在蛋白水平的表达.另收集蜡块组织SCC 50份、Bowen病20份、AK 20份及正常表皮组织10份,免疫组化检测PKD1、pPKD1-tyr463及pPKD1-ser916的表达情况.结果 正常皮肤组织、SCC、Bowen病和AK组织中PRKD1 mRNA的表达量分别为0.64±0.09、5.37±1.06、2.69±0.72和2.43±0.46,4组间差异有统计学意义(F=21.37,P＜0.05),且SCC、Bowen病和AK组织的表达水平均显著高于正常组织(P＜0.05),SCC组织又显著高于AK和Bowen病组织(均P＜ 0.05),而Bowen病与AK组织的表达量差异无统计学意义(P＞0.05).PKD1总蛋白及pPKD1-tyr463在SCC和Bowen病组织中主要表达在棘层细胞及异形细胞的细胞质和细胞膜,且阳性表达率均显著高于正常皮肤组和AK组(均P＜0.01);pPKD1-ser916仅在部分高分化SCC癌巢中少量表达,而低分化鳞癌、AK、Bowen病及正常皮肤组织中均未见表达;SCC组中PKD1阳性表达率随鳞癌病理分级的提高而增加,且PKD1与pPKD1-tyr463的表达呈正相关(rcc=0.479,P＜0.05).Western印迹检测结果与免疫组化检测结果大致相符.结论 PKD1及其磷酸化位点Tyr463可能参与复层鳞状上皮来源的皮肤肿瘤的形成和进一步发展分化,在皮肤SCC形成进程中PKD1可能通过Tyr463位点活化而发挥促进作用.     

p27、细胞周期素D_1在恶性角质形成细胞肿瘤中的表达

目的探讨恶性皮肤角质形成细胞肿瘤中 p27、细胞周期素 D1的蛋白表达与角质形成细胞肿瘤的关系。方法用免疫组化法检测肿瘤细胞 p27与细胞周期素 D1蛋白表达。结果 p27在 Bowen病和日光性角化病均为高表达,基底细胞癌和鳞状细胞癌为低表达,而且前两者的表达显著高于后两者（ P< 0.05）。细胞周期素 D1在 Bowen病、基底细胞癌及日光性角化病均无表达,而在鳞状细胞癌表达率很高。结论 p27和细胞周期素 D1蛋白表达在皮肤角质形成细胞肿瘤中的改变具有一定肿瘤特异性,可作为判断角质形成细胞肿瘤恶性程度、评估预后的指标。     

p27、细胞周期素D1在恶性角质形成细胞肿瘤中的表达

目的：探讨恶性皮肤角质形成细胞肿瘤中p27、细胞周期素D1的蛋白表达与角质形成细胞肿瘤的关系。方法：用免疫组化法检测肿瘤细胞p27与细胞周期素D1蛋白表达。结果：p27在Bowen病和日光性角化病均为高表达，基底细胞癌和麟状细胞癌为低表达，而且前两者的表达显著高于后两者（P＜0．05）。细胞周期素D1在Bowen病、基底细胞癌及日光性角化病均无表达，而在麟状细胞癌表达率很高。结论：p27和细胞周期素D1蛋白表达在皮肤角质形成细胞肿瘤中的改变具有一定肿瘤特异性，可作为判断角质形成细胞肿瘤恶性程度、评估预后的指标。     

氨基酮戊酸光动力疗法对鳞状细胞癌细胞株A431蛋白激酶D1及其磷酸化位点的影响

目的 研究氨基酮戊酸光动力疗法（ALA-PDT）对皮肤鳞状细胞癌A431细胞蛋白激酶D1（PKD1）的影响,探讨ALA-PDT诱导A431细胞凋亡的机制。方法 体外培养A431细胞,用CCK-8法检测筛选出抑制作用最强的ALA浓度和光动力照射（PDT）剂量组合。将对数期生长A431细胞随机分为不予任何干预的对照组、ALA组、PDT组及ALA-PDT组,观察4组细胞接受相应干预12、24、36、48 h后细胞增殖抑制率和增殖抑制作用最强时间点的凋亡率。反转录PCR检测ALA-PDT作用A431细胞不同时间点后各组PKD1基因mRNA的表达。Western 印迹法检测各组A431细胞中PKD1及其磷酸化位点Tyr463蛋白（pTyr463）、Ser916蛋白（pSer916）表达。结果 ALA浓度1.5 mmol/L、光照剂量2 J/cm2为最佳剂量组合。4组细胞干预12、24、36、48 h的增殖抑制率差异有统计学意义（F = 39.56,P < 0.05）。孵育24 h时：ALA-PDT组细胞增殖抑制率（46.26% ± 1.25%）高于ALA组（14.65% ± 0.33%）、PDT组（14.96% ± 0.68%）和对照组（11.98% ± 0.32%）,差异有统计学意义（均P < 0.05 ）;ALA-PDT组细胞增殖抑制率高于12 h（P < 0.05）;4组细胞凋亡率差异有统计学意义（F = 16.32,P < 0.05）,ALA-PDT组（41.92% ± 3.23%）高于对照组（4.67% ± 0.88%）、ALA组（7.02% ± 1.52%）和PDT组（8.37% ± 0.59%）,均P < 0.05。ALA-PDT作用于A431细胞后0、6、12、24、36、48 h,细胞PKD1基因mRNA的相对表达量差异有统计学意义（F = 22.24,P < 0.05）,孵育24 h mRNA相对表达量低于0、6、12 h（均P < 0.05）,与36、48 h差异无统计学意义（均P > 0.05）。4组A431细胞中pSer916表达量差异无统计学意义（F = 1.53,P > 0.05）,PKD1和pTyr463表达量差异有统计学意义（F值为10.04、8.27,均P < 0.05）,ALA-PDT组PKD1、pTyr463的表达低于对照组、ALA组、PDT组（均P < 0.05）。结论 PKD1可能参与ALA-PDT疗法诱导A431细胞凋亡的光化学反应进程,且可能是通过Tyr463位点活化促进癌变的发展。     

皮肤病中c-Jun氨基末端蛋白激酶通路的研究现况

c-Jun氨基末端蛋白激酶是丝裂原活化蛋白激酶家族的成员,调节细胞的生长、发育、增殖和分化.c-Jun氨基末端蛋白激酶的异常表达与人皮肤疾病的发生、发展密切相关.研究证实,c-Jun氨基末端蛋白激酶在多种皮肤疾病患者的真/表皮中表达过度增高及异常活化,进而导致细胞的生长、发育、增殖、分化异常以及炎症反应和细胞凋亡的发生.选择阻断c-Jun氨基末端蛋白激酶通路的异常活化可以使病情得到改善.针对c-Jun氨基末端蛋白激酶信号通路的靶向治疗已经成为目前多种皮肤病的研究热点.     

桥粒芯糖蛋白1和2在不同表皮肿瘤中的表达

目的 探讨桥粒相关蛋白与皮肤肿瘤的关系,对桥粒芯糖蛋白1和2在鳞状细胞癌、日光性角化病、角化棘皮瘤、脂溢性角化病中的表达水平进行了比较研究.方法 免疫组化染色方法.结果 桥粒芯糖蛋白1和2在正常皮肤表皮全层细胞间呈现较强的染色,鳞状细胞癌组织中表达显着减弱或完全无表达,日光性角化病表皮正常区域表达正常或下调,细胞间变区域无染色,角化棘皮瘤和脂溢性角化病表皮中表达水平与正常皮肤的表达非常近似.结论 桥粒芯糖蛋白1和2在恶性皮肤癌中表达下调,可能与皮肤肿瘤的侵袭和转移有关.     

皮肤鳞状细胞癌STAT3和MAPK磷酸化及cyclinD1蛋白表达的意义

目的探讨磷酸化信号传导和转录激活因子3(p-STAT3)、磷酸化丝裂原激活的蛋白激酶(p-MAPK)、细胞周期蛋白D1(cyc linD1)在皮肤鳞状细胞癌(SCC)的水平和意义。方法免疫组化方法观察30例SCC中p-STAT3,p-MAPK和cyc linD1蛋白的水平。结果①p-STAT3的水平在SCC中呈明显上调(P<0.001);而p-MAPK在正常皮肤和表皮良恶性肿瘤中的水平差异无统计学意义。②p-STAT3在皮肤SCC中的阳性强度与肿瘤的分化程度有关(P<0.05),阳性率与肿瘤浸润的深度有关(P<0.05),与肿瘤的大小无关。③皮肤SCC中,p-STAT3和cyc linD1的阳性强度呈正相关(P<0.05)。p-MAPK和cyc linD1的阳性强度无相关性。结论①STAT3的磷酸化可能在皮肤SCC的发生发展中起重要作用,并可作为皮肤SCC预后判断的辅助指标之一。②在皮肤SCC中,p-STAT3可能通过诱导cyc linD1过度表达,从而促使肿瘤细胞维持高增殖状态。     

PTEN基因在皮肤肿瘤中的研究进展

PTEN基因是一个抑癌基因,其缺失、突变和表达减少可存在多种皮肤肿瘤中.PTEN基因主要通过PI3K/Akt、FAK/p130途径及Shc/ras/MARK途径影响细胞的分化、凋亡、移动及信号转导等方面,参与皮肤肿瘤的发生发展,与皮肤肿瘤的恶性转化有着密切的关系.因此,PTEN基因在保持皮肤内环境稳定和抑制皮肤肿瘤的发生方面起着重要的作用.概述紫外线辐射对PTEN功能的调节及PTEN基因在多种皮肤肿瘤发生发展中的作用.     

皮肤鳞状细胞癌中Basigin/CD147表达与肿瘤进展的相关性

目的 探讨Basigin/CD147在皮肤鳞状细胞癌(鳞癌)进展中的作用及其机制.方法 运用免疫组化方法检测36例浸润和复发/转移情况不同的原发皮肤鳞状细胞癌标本中Basigin和基质金属蛋白酶(MMPs)的表达.运用免疫印迹和免疫荧光法观察Basigin在培养角质形成细胞和鳞状细胞癌细胞中的表达和定位.结果 有浸润和伴复发/转移的原发鳞癌肿瘤细胞中Basigin、MMP-1、MMP-2和MT1-MMP的表达显着增高.肿瘤细胞周围成纤维细胞中MMP-1、MMP-2和MT1-MMP的表达亦与原发鳞癌的复发/转移密切相关.肿瘤细胞膜上Basigin的表达与成纤维细胞中MMP-1、MMP-2、MMP-3和MT1-MMP的表达间显着相关.增高表达的Basigin主要分布于鳞癌细胞膜的微绒毛上.结论 鳞癌细胞膜上增高表达的Basigin/CD147可能与周围成纤维细胞作用,诱导其MMPs产生,并通过这一机制在鳞癌进展中发挥重要作用.     

与紫外线致皮肤损伤相关的部分信号通路研究进展

紫外线照射可使皮肤损伤,导致皮肤光老化或皮肤肿瘤.多个信号通路如Nrf2-Keap1-ARE、核因子κB、丝裂原活化蛋白激酶(MAPK)、磷脂酰肌醇-3激酶-丝氨酸/苏氨酸激酶-西罗莫司靶蛋白(PI3K-AKt-mTOR)信号通路等参与皮肤光老化和(或)皮肤肿瘤的发病.Nrf2信号通路在氧化应激状态下开启,有维持氧化还原平衡和参与细胞新陈代谢等作用.核因子κB信号通路的激活引起基质金属蛋白酶等水平上调,与皮肤老化和非黑素性皮肤癌有关.MAPK信号通路参与皮肤老化和皮肤肿瘤的进展.PI3K-AKt-mTOR信号通路主要与皮肤肿瘤相关.紫外线照射可诱导上述通路的活化,参与皮肤光老化或皮肤肿瘤的发生发展.对上述通路的进一步研究有望为抵御皮肤的光老化和皮肤肿瘤提供新的方法.     

Proteinase-activated receptors 1 and 2 regulate invasive behavior of human melanoma cells via activation of protein kinase D1

Recent studies have indicated an important role of proteinases and proteinase-activated receptors (PARs) in tumorigenesis. Although a role for PARs has been described in various skin tumors including melanoma, the underlying cellular mechanisms have not been understood. Recent studies have suggested PAR(1) as a regulator of melanoma cell growth and metastasis by affecting angiogenic and invasive factors. Moreover, changes in the expression patterns of PAR(1) and PAR(2) correlate with skin cancer progression, and PAR(1) is overexpressed in melanoma. Therefore, we sought to elucidate the putative role of PAR(1)- and PAR(2)-mediated signal transduction pathways during melanoma progression. Activation of both PAR(1) and PAR(2) led to rapid phosphorylation of protein kinase D1 (PKD1) in cultured WM9 melanoma cells. PKD1 is known to be involved in cell migration, integrin regulation, and intracellular vesicle transport. Downregulation of PKD1 by siRNA resulted in diminished proliferation, decreased αvβ3 integrin regulation, and secretion of pro-angiogenic chemokine IL-8 in WM9 cells. In conclusion, our results show that PAR(1) and PAR(2) are involved in WM9 cell proliferation and secretion of IL-8 by activation of PKD1. Inactivation of the PKD1 pathway may be beneficial for the inhibition of PAR-induced melanoma proliferation and for maintenance of the inflammatory tumor environment.     

Regulation of protein kinase D during differentiation and proliferation of primary mouse keratinocytes

Diseased skin often exhibits a deregulated program of the keratinocyte maturation necessary for epidermal stratification and function. Protein kinase D (PKD), a serine/threonine kinase, is expressed in proliferating keratinocytes, and PKD activation occurs in response to mitogen stimulation in other cell types. We have proposed that PKD functions as a pro-proliferative and/or anti-differentiative signal in keratinocytes and hypothesized that differentiation inducers will downmodulate PKD to allow differentiation to proceed. Thus, changes in PKD levels, autophosphorylation, and activity were analyzed upon stimulation of differentiation and proliferation in primary mouse keratinocytes. Elevated extracellular calcium and acute 12-O-tetradecanoylphorbol-13-acetate (TPA) treatments induced differentiation and triggered a downmodulation of PKD levels, autophosphorylation at serine 916, and activity. Chronic TPA treatment stimulated proliferation and resulted in a recovery of PKD levels, autophosphorylation, and activity. Immunohistochemical analysis demonstrated PKD localization predominantly in the proliferative basal layer of mouse epidermis. Co-expression studies revealed a pro-proliferative, anti-differentiative effect of PKD on keratinocyte maturation as monitored by increased and decreased promoter activities of keratin 5, a proliferative marker, and involucrin, a differentiative marker, respectively. This work describes the inverse regulation of PKD during keratinocyte differentiation and proliferation and the pro-proliferative/anti-differentiative effects of PKD co-expression on keratinocyte maturation.     

Role of calcium in polycystic kidney disease: From signaling to pathology

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited monogenic kidney disease. Characterized by the development and growth of cysts that cause progressive kidney enlargement, it ultimately leads to end-stage renal disease. Approximately 85% of ADPKD cases are caused by mutations in the PKD1 gene, while mutations in the PKD2 gene account for the remaining 15% of cases. The PKD1 gene encodes for polycystin-1 (PC1), a large multi-functional membrane receptor protein able to regulate ion channel complexes, whereas polycystin-2 (PC2), encoded by the PKD2 gene, is an integral membrane protein that functions as a calcium-permeable cation channel, located mainly in the endoplasmic reticulum (ER). In the primary cilia of the epithelial cells, PC1 interacts with PC2 to form a polycystin complex that acts as a mechanosensor, regulating signaling pathways involved in the differentiation of kidney tubular epithelial cells. Despite progress in understanding the function of these proteins, the molecular mechanisms associated with the pathogenesis of ADPKD remain unclear. In this review we discuss how an imbalance between functional PC1 and PC2 proteins may disrupt calcium channel activities in the cilium, plasma membrane and ER, thereby altering intracellular calcium signaling and leading to the aberrant cell proliferation and apoptosis associated with the development and growth of renal cysts. Research in this field could lead to the discovery of new molecules able to rebalance intracellular calcium, thereby normalizing cell proliferation and reducing kidney cyst progression.     

Knockdown of PKD1 in normal human epidermal keratinocytes increases mRNA expression of keratin 10 and involucrin: early markers of keratinocyte differentiation

Subconfluent normal human keratinocytes exhibit autonomous (autocrine growth factor driven) proliferation and express the specific markers for keratinocyte proliferation K5 (keratin 5) and K14 (keratin 14). Utilizing this model the effects of PKD1 (Protein kinase D1) knockdown on activation of differentiation was studied. siRNA approach was applied to achieve specific knockdown of PKD1 and the mRNA levels of different keratinocyte markers—K14 and PCNA (markers of basal proliferating keratinocytes), involucrin and K10 (early differentiation markers) were analyzed. Treatment of cultured keratinocytes with siRNA for PKD1 resulted in reduction of mRNA levels of PKD1, altered cell phenotype and promotion of keratinocyte differentiation, demonstrated by increased expression of involucrin and K10 mRNAs. No significant changes in K14 mRNA expression levels were detected, but the expression of PCNA mRNA was markedly diminished. This study was the first to show that mRNA expression of PKD1 in subconfluent normal human keratinocytes is very low, the PKD1 mRNA levels were more than 8-fold lower than the same ones in hTert keratinocytes. These findings suggest antidifferentiative role of PKD1 in normal human keratinocytes, contrary to the prodiferentiative role of PKD1 in human hTert keratinocytes. We came to the conclusion that there are differences between transduction pathways involving PKD1 in primary human keratinocyte cultures and these in immortalized hTert keratinocytes.     

Cytokeratin expression patterns in multiple infundibulocystic basal cell carcinoma

Infundibulocystic basal cell carcinoma (IBCC) is a variant of basal cell carcinoma. Sporadic cases usually represent a solitary tumor and multiple IBCC is rare. There have been no reports in which the tumor differentiation is characterized immunohistochemically. We report a case of multiple IBCC which developed on a patient's scalp by performing histopathological and immunohistochemical examinations, using monoclonal antibodies against cytokeratins (CKs). A 76‐year‐old female had noticed multiple small papules on her scalp. She noticed that the tumors were growing when she underwent systemic chemotherapy for metastatic lung cancer. Routine histopathological specimens from skin biopsies revealed findings typical of IBCC. The tumor cells expressed CK14 and CK17. However, CK1 and CK10 were expressed only in a few cells in the inner area of the tumors. The present case is unique in two points. First, multiple tumors developed on the patient's scalp during the systemic chemotherapy for the lung cancer. Second, the tumor showed CK expression patterns characteristic to infundibular and trichilemmal epithelia. Hida T, Saga K, Kimura T. Cytokeratin expression patterns in multiple infundibulocystic basal cell carcinoma.     

Constitutive renal Rel/nuclear factor-&kappa;B expression in Lewis polycystic kidney disease rats

AIM: To determine the temporal expression and pattern of Rel/nuclear factor (NF)-κB proteins in renal tissue in polycystic kidney disease (PKD). METHODS: The renal expression of Rel/NF-κB proteins was determined by immunohistochemistry, immunofluorescence and immunoblot analysis in Lewis polycystic kidney rats (LPK, a genetic ortholog of human nephronopthsis-9) from postnatal weeks 3 to 20. At each timepoint, renal disease progression and the mRNA expression of NF-κB-dependent genes (TNFα and CCL2) were determined. NF-κB was also histologically assessed in human PKD tissue. RESULTS: Progressive kidney enlargement in LPK rats was accompanied by increased renal cell proliferation and interstitial monocyte accumulation (peaking at weeks 3 and 10 respectively), and progressive interstitial fibrosis (with α smooth muscle actin and Sirius Red deposition significantly increased compared to Lewis kidneys from weeks 3 to 6 onwards). Rel/NF-κB proteins (phosphorylated-p105, p65, p50, c-Rel and RelB) were expressed in cystic epithelial cells (CECs) of LPK kidneys as early as postnatal week 3 and sustained until late-stage disease at week 20. From weeks 10 to 20, nuclear p65, p50, RelB and cytoplasmic IκBα protein levels, and TNFα and CCL2 expression, were upregulated in LPK compared to Lewis kidneys. NF-κB proteins were consistently expressed in CECs of human PKD. The DNA damage marker γ-H2AX was also identified in the CECs of LPK and human polycystic kidneys. CONCLUSION: Several NF-κB proteins are consistently expressed in CECs in human and experimental PKD. These data suggest that the upregulation of both the canonical and non-canonical pathways of NF-κB signaling may be a constitutive and early pathological feature of cystic renal diseases.     

Protein kinase D distribution in normal human epidermis, basal cell carcinoma and psoriasis

BACKGROUND: Keratinocytes undergo a defined programme of proliferation and differentiation during normal stratification of the epidermis. Anomalies in the signalling pathways controlling this process probably contribute to the pathogenesis of hyperproliferative dermatological diseases, including psoriasis and basal cell carcinoma (BCC). We have previously proposed that protein kinase D (PKD) is a proproliferative signalling enzyme in keratinocytes and have speculated that abnormalities in its levels or regulation may contribute to hyperproliferative disorders of the skin. OBJECTIVES: To determine if hyperproliferative human skin disorders are characterized by abnormal protein expression or distribution of PKD, normal human epidermis was compared with BCC and uninvolved and involved psoriatic epidermis. METHODS: To examine protein expression, immunohistochemical analysis of human samples and Western blotting of neoplastic mouse keratinocytes was performed. Western analysis of neoplastic mouse cells using a phosphospecific PKD antibody allowed estimation of PKD activation status. RESULTS: Normal human epidermis demonstrated predominant PKD protein expression in the stratum basalis, the proliferative epidermal compartment, with decreased relative expression throughout the suprabasal strata. Uninvolved psoriatic skin showed a similar pattern, but in contrast, psoriatic lesions demonstrated a diffuse distribution of PKD staining throughout all strata. The majority of BCCs examined showed significant PKD protein levels and, in those biopsies in which the levels could be compared, elevated PKD levels relative to normal epidermis. PKD levels and activation status were also increased in a neoplastic mouse keratinocyte cell line. CONCLUSIONS: PKD was elevated or misdistributed in the hyperproliferative human skin disorders, BCC and psoriasis, as well as neoplastic mouse keratinocytes. We speculate that PKD exerts proproliferative and/or antidifferentiative effects in the epidermis, and that anomalous distribution and/or activation of PKD may be involved in precipitating or sustaining the disease process in BCC and psoriasis.     

Troglitazone inhibits cyclin D1 expression and cell cycling independently of PPARgamma in normal mouse skin keratinocytes

Troglitazone is one of the thiazolidinedione (TZD) class of anti-diabetic drugs and a ligand for peroxisome proliferator-activated receptor gamma (PPARgamma). Troglitazone and other PPARgamma ligands have been shown to inhibit cell proliferation and induce cell cycle arrest in a variety of cancer cells, and have been considered as potential tumor preventive and tumor therapeutic agents. Little is known, however, about how normal or initiated cells respond to these agents during mouse skin carcinogenesis. We report here that troglitazone and another TZD, ciglitazone, dramatically inhibited mitogen-induced cellular proliferation in normal mouse skin primary keratinocytes and in the C50 keratinocyte cell line. This was accompanied by induction of cell cycle G1 phase arrest and suppression of cyclin D1, cdk4, and cdk2 expression. Troglitazone suppressed cyclin D1 expression at multiple levels. In addition, we demonstrated that PPARgamma was not expressed at functional levels in cultured mouse skin keratinocytes, and that the inhibitory effects of troglitazone on cellular proliferation and cyclin D1 expression in these cells were PPARgamma-independent. Given the important role of keratinocyte proliferation in skin carcinogenesis, our data suggest that TZD may be useful tumor preventive agents in skin.