皮膚病中蛋白激酶D1研究進(jìn)展

顧 靜 劉保國(guó) 周 萌 張海芳

?

·綜述·

皮膚病中蛋白激酶D1研究進(jìn)展

顧 靜1劉保國(guó)2周 萌1張海芳3

蛋白激酶D1是一種在體內(nèi)多個(gè)重要器官?gòu)V泛表達(dá)的鈣離子/鈣調(diào)蛋白依賴性的絲氨酸/蘇氨酸蛋白激酶，參與多種重要的生理和病理活動(dòng)，在許多腫瘤組織中表達(dá)異常。PKD1促進(jìn)角質(zhì)形成細(xì)胞增殖,抑制其分化，對(duì)表皮傷口愈合及腫瘤的形成具有促進(jìn)作用，與UVB、ROS等影響皮膚疾病發(fā)生發(fā)展的重要因素之間都有密切聯(lián)系。因此PKD1可能成為治療某些皮膚病甚至皮膚腫瘤更有效的分子靶點(diǎn)。

蛋白激酶D1； 角質(zhì)形成細(xì)胞； 皮膚疾病

蛋白激酶D(protein kinase D,PKD)是一種鈣離子/鈣調(diào)蛋白依賴性的絲氨酸/蘇氨酸蛋白激酶。首先由1994年Rozengurt等[1]發(fā)現(xiàn)，曾被認(rèn)為是蛋白激酶C(PKC)家族成員(protein kinase mu,PKCμ)之一[2]，由于其激酶結(jié)構(gòu)域與鈣離子/鈣調(diào)蛋白依賴性激酶(Ca2+/calmodulin-dependent kinase,CAMK)家族的同源性高于PKC家族,具有PKC激酶家族成員所沒有的疏水端和一個(gè)特異性的底物同源結(jié)構(gòu)域(pleckstrin-homology domain,PH)，故現(xiàn)已歸為CAMK激酶家族[3]。目前已確定三個(gè)PKD亞型，分別為PKD1(PKCμ)、PKD2和PKD3(PKCυ)，三者在結(jié)構(gòu)上存在相似之處，能被相同的刺激因子激活但卻有著不同的功能，因?yàn)樗麄兙哂胁煌谋磉_(dá)水平和組織特異性，且彼此之間存在著復(fù)雜的相互作用[4]。早期文獻(xiàn)中PKD僅指PKD1，目前關(guān)注最多的也是PKD1，已證明其參與人體多種生理和病理活動(dòng)，與皮膚疾病的關(guān)系也越來越多的引起學(xué)者們的興趣[5,6]。

1 PKD1概述

1.1 PKD1在細(xì)胞內(nèi)分布在靜息狀態(tài)下的細(xì)胞中，大部分PKD1定位于細(xì)胞質(zhì)，少部分存在于高爾基體和細(xì)胞核，在一些特異細(xì)胞中還可分布在線粒體或分泌顆粒中。PKD1活化后可移位至不同的細(xì)胞結(jié)構(gòu)中，Ser744/748位點(diǎn)的磷酸化可能參與PKD1在細(xì)胞中分布位置的調(diào)節(jié)。其富含鋅指結(jié)構(gòu)和底物同源區(qū)的N段結(jié)構(gòu)域使其激酶活性處于很低的狀態(tài)，在受到佛波酯、G蛋白偶聯(lián)受體激動(dòng)劑、生長(zhǎng)因子等刺激后立即活化,并被募集到細(xì)胞膜、細(xì)胞核或高爾基體等位置[7,8]。

1.2 PKD1在組織內(nèi)的表達(dá)PKD1基因(又稱PRKD1)位于人染色體14qll位置，在體內(nèi)多個(gè)重要器官如腦、心臟、肺中廣泛表達(dá)，在多種腫瘤中表達(dá)異常，與腫瘤的發(fā)生及侵襲轉(zhuǎn)移密切相關(guān)[9-11]，如在前列腺癌、乳腺癌、非小細(xì)胞肺癌、胃癌和結(jié)直腸癌中PKD1顯著下調(diào)[11-14],而高表達(dá)的PKD1在胰腺癌、皮膚基底細(xì)胞癌及黑素瘤的發(fā)生和轉(zhuǎn)移中起重要作用[15-17]。

1.3 PKD1參與調(diào)控細(xì)胞功能和多條細(xì)胞內(nèi)信號(hào)傳導(dǎo)通路，其中與皮膚病關(guān)系較密切的主要有：① 氧化應(yīng)激條件下PKD1活化，通過NF-κB途徑幫助表皮角質(zhì)形成細(xì)胞存活。表皮角質(zhì)形成細(xì)胞受到氧化應(yīng)激損傷后，會(huì)引起線粒體上PKD1活化，從而激活 PKD1-IKK-NF-κB信號(hào)通路。NF-κB轉(zhuǎn)錄因子將誘導(dǎo)超氧化物歧化酶2(superoxide dismutase2,SOD2)基因的表達(dá),繼而保護(hù)細(xì)胞避免凋亡[18]。② PKD1通過磷酸化組蛋白去乙酰化酶HDAC，影響HDAC對(duì)基因表達(dá)的調(diào)控作用進(jìn)而調(diào)節(jié)有絲分裂信號(hào)。在以皮膚色素沉著、脫失、掌跖角化及癌變?yōu)樘卣鞯纳橹卸静∽冎锌赡馨l(fā)揮一定的作用[19,20]。③ PKD1通過 RINI (—種競(jìng)爭(zhēng)性Ras效應(yīng)蛋白)誘導(dǎo)ERK通路的持續(xù)激活，誘導(dǎo)原癌基因c-Fos的聚集促進(jìn)細(xì)胞DNA合成，調(diào)節(jié)細(xì)胞的有絲分裂[21]。

1.4 PKD1與皮膚病相關(guān)的主要激活途徑：① 通過催化域上的兩個(gè)磷酸化位點(diǎn)Ser744/748激活，即PKC(磷脂酶C)依賴性PLC-PKC-PKD1途徑[22]。通過PKC使PKD1分子的Ser744位點(diǎn)磷酸化及Ser748位點(diǎn)自身磷酸化，從而突破PH結(jié)構(gòu)域的抑制而使PKD1處于穩(wěn)定的活化狀態(tài)。其后常常伴隨Ser916位點(diǎn)的磷酸化，標(biāo)志著PKD1的完全活化，有研究表明皮膚黑素瘤細(xì)胞中PKD1便是以此途徑活化[17，23]。② 通過Tyr463位點(diǎn)磷酸化激活，在氧化應(yīng)激等條件下酪氨酸激酶的活性增強(qiáng)，活化的Src作用于PKD1分子的PH結(jié)構(gòu)域，使Tyr463位點(diǎn)發(fā)生磷酸化，結(jié)合目前的研究我們考慮PKD1在皮膚非黑素瘤中可能是通過該途徑活化的[24]。

2 PKD1與某些皮膚病的相關(guān)性

2.1 PKD1與角質(zhì)形成細(xì)胞 角質(zhì)形成細(xì)胞是表皮組織最重要的功能細(xì)胞，其增殖和分化失衡可致皮膚防御功能損傷及多種皮膚疾病發(fā)生。PKD1信號(hào)通路對(duì)維持表皮平衡具有至關(guān)重要的作用[25]。通過RNA干擾技術(shù)降低PKD1的表達(dá)到原有水平的20%，足以阻止角質(zhì)形成細(xì)胞的增殖和逆轉(zhuǎn)分化[26]。Choudhary等研究證實(shí)PKD1的缺失可導(dǎo)致各種分化標(biāo)記及相關(guān)mRNA表達(dá)顯著增加[27]。Rashel等發(fā)現(xiàn)PKD1表達(dá)的多少并不影響表皮組織的正常發(fā)育和穩(wěn)態(tài)，然而有趣的是在表皮損傷的情況下，PKD1的缺失可致傷口周圍的角質(zhì)形成細(xì)胞增殖和遷移明顯較正常組減緩，出現(xiàn)傷口再上皮化延遲[28]。近期他們又提出不同的PKD亞型在人角質(zhì)形成細(xì)胞中具有不同的作用，PKD2缺失增加角質(zhì)形成細(xì)胞的增殖潛能，PKD3缺失卻可導(dǎo)致增殖能力缺陷使得克隆形成及組織再生能力減少，而PKD1對(duì)角質(zhì)形成細(xì)胞的作用可能相對(duì)較弱[29]。這和美國(guó)佐治亞醫(yī)學(xué)研究所及我們前期所得實(shí)驗(yàn)結(jié)果相悖，PKD1在人表皮組織的真實(shí)表達(dá)情況及其與皮膚病的關(guān)系有待進(jìn)一步研究。

2.2 PKD1參與皮膚腫瘤形成 早在2005年Ristich 等利用免疫組化證實(shí)了PKD1在正常皮膚，銀屑病和基底細(xì)胞癌中的表達(dá)，而初步推測(cè)其在增殖性皮膚病如銀屑病和皮膚腫瘤中可能發(fā)揮作用[16]。Rashel等選用PKD1基因敲除的小鼠和正常小鼠對(duì)比進(jìn)行二階法化學(xué)誘癌實(shí)驗(yàn)，結(jié)果顯示PKD1敲除組小鼠成瘤率及腫瘤惡性程度都明顯低于正常對(duì)照組。說明PKD1的缺失可抑制腫瘤形成，進(jìn)而推測(cè)PKD1在皮膚腫瘤的形成過程中發(fā)揮重要作用[28，30]。Chiou等研究證實(shí)在皮膚腫瘤干細(xì)胞標(biāo)記CD34陽(yáng)性的細(xì)胞系和皮膚腫瘤中通過抑制PKD1相關(guān)的信號(hào)通路可有效預(yù)防皮膚腫瘤發(fā)生[31]。Kempkes等在黑素瘤細(xì)胞下調(diào)PKD1的表達(dá)結(jié)果發(fā)現(xiàn)腫瘤細(xì)胞增殖明顯下降，他們認(rèn)為PKD1相關(guān)通路的失活可能有助于抑制黑素瘤細(xì)胞的增殖及對(duì)腫瘤微環(huán)境的維護(hù)[32]。隨著對(duì)PKD1與皮膚腫瘤發(fā)生發(fā)展關(guān)系認(rèn)識(shí)的逐漸深入，越來越多的證據(jù)顯示PKD1有望成為皮膚腫瘤治療的新靶點(diǎn)。

2.3 PKD1與紫外線 紫外線與皮膚健康狀況及某些皮膚病變的發(fā)生關(guān)系極為密切，皮膚中的角質(zhì)形成細(xì)胞是UV照射的重要靶點(diǎn)。慢性UV輻射可導(dǎo)致皮膚組織中復(fù)雜的細(xì)胞內(nèi)事件和分子反應(yīng)，最終可導(dǎo)致皮膚衰老甚至惡性腫瘤的形成。研究發(fā)現(xiàn)UVB的致癌作用較UVA更強(qiáng),并可以促使ROS的產(chǎn)生[33,34]。UVB殺傷角質(zhì)形成細(xì)胞造成其凋亡的過程中同時(shí)激活細(xì)胞內(nèi)的PKD1(這種PKD1的活化形式可能是通過絲氨酸家族中的酪氨酸激酶實(shí)現(xiàn)的)，活化的PKD1有益于減少遭受低水平UVB損傷細(xì)胞的過度凋亡(低水平UVB照射引起的微小DNA損傷可以修復(fù))[24]。如果PKD1有助于已經(jīng)遭受了紫外線引起的DNA損傷細(xì)胞的存活，這些角質(zhì)形成細(xì)胞的DNA突變可能繼續(xù)擴(kuò)散，甚至形成皮膚腫瘤。

2.4 PKD1與活性氧簇(reactive oxygen species,ROS)是生物體內(nèi)細(xì)胞在有氧代謝過程中產(chǎn)生的含氧及氧代謝產(chǎn)物的一類具有高生物活性的物質(zhì)，ROS的產(chǎn)生及氧化還原狀態(tài)的調(diào)節(jié)是皮膚腫瘤細(xì)胞中常見的生化反應(yīng),低水平的ROS參與腫瘤細(xì)胞的生長(zhǎng)，ROS水平增高到一定程度轉(zhuǎn)而抑制腫瘤的發(fā)展，多數(shù)化療藥物也是通過抑制腫瘤細(xì)胞的抗氧化防御體系來誘導(dǎo)細(xì)胞凋亡[35]。Zhang等相關(guān)研究揭示PKD1參與線粒體去極化，是調(diào)節(jié)活性氧產(chǎn)生閾值的關(guān)鍵因素，PKD1出現(xiàn)在腫瘤生長(zhǎng)的特定階段，在細(xì)胞老化及營(yíng)養(yǎng)缺乏方面發(fā)揮重要功能[36]。UVB誘導(dǎo)角質(zhì)形成細(xì)胞凋亡依賴于細(xì)胞的氧化還原狀態(tài)。長(zhǎng)期反復(fù)的UV刺激帶來的慢性損傷可能導(dǎo)致細(xì)胞修復(fù)功能受損從而產(chǎn)生光老化、光致癌。因此對(duì)PKD1與角質(zhì)形成細(xì)胞氧化應(yīng)激反應(yīng)的研究對(duì)明確UV對(duì)皮膚損害的機(jī)理具有非常重要的意義。而目前運(yùn)用于多種皮膚病的光動(dòng)力療法中，光敏劑進(jìn)入組織及細(xì)胞被特定波長(zhǎng)的光照射后吸收能量轉(zhuǎn)為激發(fā)態(tài)，隨后與底物作用產(chǎn)生單線態(tài)氧及ROS作為細(xì)胞毒性物質(zhì)啟動(dòng)對(duì)腫瘤細(xì)胞的直接及間接殺傷[37]。在這個(gè)過程中PKD1是否發(fā)揮作用，發(fā)揮怎樣的作用，值得我們進(jìn)一步探究。

3 展望

PKD1在皮膚病中發(fā)揮的具體作用目前尚無定論，但多數(shù)研究者認(rèn)為PKD1促進(jìn)角質(zhì)形成細(xì)胞的增殖，抑制其分化，且與佛波酯、UVB、ROS等影響皮膚疾病的重要因素間存在密切的聯(lián)系，可能在某些皮膚病甚至皮膚腫瘤的形成中發(fā)揮重要的作用。然而PKD1能否成為增殖性皮膚病或皮膚腫瘤治療中的新靶點(diǎn)，尚需更深入的探究。

[1] Valverde AM, Sinnett-Smith J, Van Lint J, et al. Molecular cloning and characterization of protein kinase D: a target for diacylglycerol and phorbol esters with a distinctive catalytic domain[J]. Proc Natl Acad Sci USA,1994,91(18):8572-8576.

[2] Johannes FJ, Prestle J, Eis S, et al. PKCu is a novel, atypical member of the protein kinase C family[J]. J Biol Chem,1994,269(8):6140-6148.

[3] Rykx A, De Kimpe L, Mikhalap S, et al. Protein kinase D: a family affair[J]. FEBS Lett,2003,546(1):81-86.

[4] Fu Y, Rubin CS. Protein kinase D: coupling extracellular stimuli to the regulation of cell physiology[J]. EMBO Rep,2011,12(8):785-796.

[5] Ellwanger K, Hausser A. Physiological functions of protein kinase D in vivo[J]. IUBMB Life,2013,65(2):98-107.

[6] Rozengurt E. Protein kinase D signaling: multiple biological functions in health and disease[J]. Physiology (Bethesda),2011,26(1):23-33.

[7] Van Lint JV, Sinnett-Smith J, Rozengurt E. Expression and characterization of PKD, a phorbol ester and diacylglycerol-stimulated serine protein kinase[J]. J Biol Chem,1995,270(3):1455-1461.

[8] Zugaza JL, Waldron RT, Sinnett-Smith J, et al. Bombesin, vasopressin, endothelin, bradykinin, and platelet-derived growth factor rapidly activate protein kinase D through a protein kinase C-dependent signal transduction pathway[J]. J Biol Chem,1997,272(38):23952-23960.

[9] Li G, Wang Y. Protein kinase D: a new player among the signaling proteins that regulate functions in the nervous system[J]. Neurosci Bull,2014,30(3):497-504.

[10] Eiseler T, K?hler C, Nimmagadda SC, et al. Protein kinase D1 mediates anchorage-dependent and -independent growth of tumor cells via the zinc finger transcription factor Snail1[J]. J Biol Chem,2012,287(39):32367-32380.

[11] Tandon M, Salamoun JM, Carder EJ, et al. SD-208, a novel protein kinase D inhibitor, blocks prostate cancer cell proliferation and tumor growth in vivo by inducing G2/M cell cycle arrest[J]. PLoS One,2015,10(3):e0119346.

[12] Karam M, Legay C, Auclair C, et al. Protein kinase D1 stimulates proliferation and enhances tumorigenesis of MCF-7 human breast cancer cells through a MEK/ERK-dependent signaling pathway[J]. Exp Cell Res,2012,318(5):558-569.

[13] Wei N, Chu E, Wipf P, et al. Protein kinase d as a potential chemotherapeutic target for colorectal cancer[J]. Mol Cancer Ther,2014,13(5):1130-1141.

[14] Sundram V, Ganju A, Hughes JE, et al. Protein kinase D1 attenuates tumorigenesis in colon cancer by modulating β-catenin/T cell factor activity[J]. Oncotarget,2014,5(16):6867-6884.

[15] Liou GY, Storz P. Protein kinase D enzymes: novel kinase targets in pancreatic cancer[J]. Expert Rev Gastroenterol Hepatol,2015,9(9):1143-1146.

[16] Ristich VL, Bowman PH, Dodd ME, et al. Protein kinase D distribution in normal human epidermis, basal cell carcinoma and psoriasis[J]. Br J Dermatol,2006,154(4):586-593.

[17] Kempkes C, Rattenholl A, Buddenkotte J, et al. Proteinase-activated receptors 1 and 2 regulate invasive behavior of human melanoma cells via activation of protein kinase D1[J]. J Invest Dermatol,2012,132(2):375-384.

[18] Storz P, Toker A. Protein kinase D mediates a stress-induced NF-kappaB activation and survival pathway[J]. EMBO J,2003,22(1):109-120.

[19] Gray SG, Teh BT. Histone acetylation/deacetylation and cancer: an "open" and "shut" case[J]. Curr Mol Med,2001,1(4):401-429.

[20] 潘雪莉,張愛華,黃曉欣.HDAC1在燃煤污染型砷中毒患者血液及皮膚組織中的轉(zhuǎn)錄及表達(dá)[J].環(huán)境與職業(yè)醫(yī)學(xué),2010,5:270-274.

[21] Wang P, Han L, Shen H, et al. Protein kinase D1 is essential for Ras-induced senescence and tumor suppression by regulating senescence-associated inflammation[J]. Proc Natl Acad Sci USA,2014,111(21):7683-7688.

[22] Waldron RT, Rey O, Iglesias T, et al. Activation loop Ser744 and Ser748 in protein kinase D are transphosphorylated in vivo[J]. J Biol Chem,2001,276(35):32606-32615.

[23] Jensen DD, Zhao P, Jimenez-Vargas NN, et al. Protein kinase D and Gβγ subunits mediate agonist-evoked translocation of protease-activated receptor-2 from the golgi apparatus to the plasma membrane[J]. J Biol Chem,2016,291(21):11285-11299.

[24] Arun SN, Kaddour-Djebbar I, Shapiro BA, et al. Ultraviolet B irradiation and activation of protein kinase D in primary mouse epidermal keratinocytes[J]. Oncogene,2011,30(13):1586-1596.

[25] Bollag WB, Dodd ME, Shapiro BA. Protein kinase D and keratinocyte proliferation[J]. Drug News Perspect,2004,17(2):117-126.

[26] Rennecke J, Rehberger PA, Fürstenberger G, et al. Protein-kinase-Cmu expression correlates with enhanced keratinocyte proliferation in normal and neoplastic mouse epidermis and in cell culture[J]. Int J Cancer,1999,80(1):98-103.

[27] Choudhary V, Olala LO, Kaddour-Djebbar I, et al. Protein kinase D1 deficiency promotes differentiation in epidermal keratinocytes[J]. J Dermatol Sci,2014,76(3):186-195.

[28] Rashel M, Alston N, Ghazizadeh S. Protein kinase D1 has a key role in wound healing and skin carcinogenesis[J]. J Invest Dermatol,2014,134(4):902-909.

[29] Ryvkin V, Rashel M, Gaddapara T, et al. Opposing growth regulatory roles of protein kinase D isoforms in human keratinocytes[J]. J Biol Chem,2015,290(17):11199-11208.

[30] Nakajima J, Nakae D, Yasukawa K. Structure-dependent inhibitory effects of synthetic cannabinoids against 12-O-tetradecanoylphorbol-13-acetate-induced inflammation and skin tumour promotion in mice[J]. J Pharm Pharmacol,2013,65(8):1223-1230.

[31] Chiou YS, Sang S, Cheng KH, et al. Peracetylated (-)-epigallocatechin-3-gallate (AcEGCG) potently prevents skin carcinogenesis by suppressing the PKD1-dependent signaling pathway in CD34+ skin stem cells and skin tumors[J]. Carcinogenesis,2013,34(6):1315-1322.

[32] Kempkes C, Rattenholl A, Buddenkotte J, et al. Proteinase-activated receptors 1 and 2 regulate invasive behavior of human melanoma cells via activation of protein kinase D1[J]. J Invest Dermatol,2012,132(2):375-384.

[33] Marrot L, Meunier JR. Skin DNA photodamage and its biological consequences[J]. J Am Acad Dermatol,2008,58(5 Suppl 2):S139-148.

[34] Rünger TM. How different wavelengths of the ultraviolet spectrum contribute to skin carcinogenesis: the role of cellular damage responses[J]. J Invest Dermato,2007,127(9):2103-2105.

[35] Yang Y, Karakhanova S, Hartwig W, et al. Mitochondria and mitochondrial ROS in cancer: novel targets for anticancer therapy[J]. J Cell Physiol,2016,231(12):2570-2581.

[36] Zhang T, Sell P, Braun U, et al. PKD1 protein is involved in reactive oxygen species-mediated mitochondrial depolarization in cooperation with protein kinase Cδ (PKCδ)[J]. J Biol Chem,2015,290(16):10472-10485.

[37] Soares HT, Campos JR, Gomes-da-Silva LC, et al. Pro-oxidant and antioxidant effects in photodynamic therapy: cells recognize that not all exogenous ROS are alike[J]. Chembiochem,2016,17(9):836-842.

(收稿：2016-07-01 修回：2016-07-08)

Update of protein kinase D1 in dermatosis

GUJing1,LIUBaoguo2,ZHOUMeng1,ZHANGHaifang3.

1.InstituteofPostgraduateDepartmentofChengdeMedicalCollege,Chengde067000,China; 2.DepartmentofDermatology,theoffiliatedHospitalofHebeiEngineeringUniversity,Handan056000,China;3.HandanCommandCenterofEmergencyRessue,Handan056000,China

LIUBaoguo,E-mail:lbg66@163.com

Protein kinase D1 (PKD1) is one calcium-dependent serine/threonine kinases extensively expressed in multiple important organs and it is involved in multiple physiological processes and various pathological conditions and abnormally expressed in tumor tissues. PKD1 promotes the proliferation and inhibits the differentiation of the keratinocytes, which play an important role in wound healing and tumor development. There is a close correlation between PKD1 and ultraviolet B, ROS, in terms of the development of skin diseases. Therefore, PKD1 may be a more effective target in the treatment of some dermatoses.

protein kinase D1; keratinocytes; dermatosis

河北省2016年度科技支撐計(jì)劃項(xiàng)目(編號(hào)：16277724D) 河北省政府資助臨床醫(yī)學(xué)優(yōu)秀人才培養(yǎng)項(xiàng)目(編號(hào)：361037)

1承德醫(yī)學(xué)院研究生學(xué)院，承德，067000 2河北工程大學(xué)附屬醫(yī)院皮膚科，邯鄲，056000 3邯鄲市緊急救援指揮中心，河北邯鄲，056000

劉保國(guó)，E-mail：lbg66@163.com