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.     

Effects of extracellular calcium on the growth-differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes

Abstract:  The keratinocyte growth and differentiation switch, tightly regulated by several mechanisms, is generally associated with decreased proliferation, cell cycle arrest in G0/G1 phase and expression of epidermal differentiation markers, such as keratin 1 (K1), keratin 10 (K10) and involucrin. In vitro , the spontaneously immortalized human keratinocyte cell line HaCaT is often used as a model to study keratinocyte functions. Comparative differentiation studies between HaCaT cells and normal human keratinocytes (NHK) over an extended time-period have rarely been reported. Therefore, we studied their switch from a proliferating to a differentiated state over 13 days. As culture conditions involved changes in cellular responses, cells were cultured in a specific medium for keratinocyte growth and differentiation was induced by increasing extracellular calcium concentration from 0.09 to 1.2 m m . In NHK, addition of calcium-induced morphological changes and concomitant decreased proliferation. For HaCaT cells, calcium addition resulted in morphological changes, but in an unexpected manner, cells were more proliferative than when cultured at low calcium levels. HaCaT cell hyperproliferation correlated with cell cycle analysis, showing an accumulation in S/G2-M phases. Furthermore, RT-PCR and western blot analysis revealed a delay in the expression of the differentiation markers K1, K10 and involucrin in HaCaT cells compared with NHK. In conclusion, even though calcium-induced differentiation was not associated with a decreased cell proliferation, HaCaT cells conserved properties characteristic of differentiation.     

Detecting expression of keratins 8/18 in human HaCaT keratinocytes

Differences in treatment solution affect the efficiency of keratin extraction in cultured human squamous cell carcinomas, malignant melanomas, and melanocytes. Using an aqueous solution that is excellent for cultured cells, we focused this study on the expression of keratin subunits in the spontaneously immortalized human keratinocyte cell line HaCaT. We extracted several keratin (K) subunits, namely K4, K7, K8, K15, K17, and K18, and ATP synthase alpha-chain, in addition to those previously reported by Boukamp et al. (J Cell Biol 1988;106:761-771) in human HaCaT keratinocytes. In particular, K8 and K18 subunits, which are related to tumorigenesis, may be very important subunits within the specificities of immortalized HaCaT cells. Vimentin, which is frequently co-expressed in cultured epithelial cell lines, was not expressed.     

IL‐1 signalling determines the fate of skin grafts expressing non‐self protein in keratinocytes

Please cite this paper as: IL‐1 signalling determines the fate of skin grafts expressing non‐self protein in keratinocytes. Experimental Dermatology 2010; 19 : 723–729. Abstract: Although IL‐1 is a known inflammatory cytokine during pathogen infection, the role of IL‐1 in skin graft rejection, particularly where foreign antigen is expressed exclusively in keratinocytes, is less understood. Here, we use a syngeneic skin graft system, where antigens are expressed in epithelial cells via either a keratin 14 or keratin 5 promoter, to explore the role of IL‐1 in graft rejection and induction of epithelial antigen‐specific effector CD8⁺ T‐cell function. Keratin 5 ovalbumin (K5mOVA) transgenic skin grafts destined for rejection demonstrated increased expression of IL‐1β and its receptors compared to K14 HPV16 E7 transgenic grafts that do not reject spontaneously. Rejection of OVA grafts lacking the IL‐1 receptor (IL‐1R1) was delayed and associated with decreased numbers of antigen‐specific CD8 T cells. In contrast, K14E7 grafts survived on immunocompetent, syngeneic recipients with decreased graft levels of IL‐1β and IL‐1R1 and 2. However, in the absence of the IL‐1 receptor antagonist, IL‐1Ra, skin grafts were spontaneously rejected and an E7‐specific CD8 T‐cell response was primed. Thus, expression of the HPV16E7 oncoprotein in epithelial cells prevents IL‐1β‐associated skin graft rejection and induction of antigen‐specific CD8 T‐cell responses. Enhancing IL‐1β signalling, via blocking of the IL‐1 receptor antagonist, may represent an alternative strategy for treatment of HPV16E7‐associated cancers.     

CHILD syndrome: lack of expression of epidermal differentiation markers in lesional ichthyotic skin.

Congenital hemidysplasia with ichthyosiform erythroderma and limb defects (CHILD) syndrome is a rare genetic disorder. The epidermal abnormalities associated with the unilateral ichthyosis have previously been examined only by morphology. In order to describe these abnormalities more completely we analyzed the expression of markers of epidermal differentiation (keratins and filaggrin), grew keratinocytes in culture, and correlated the results with ultrastructural features. Expression of all differentiation markers was significantly reduced or absent, whereas keratins K5 and K14 and keratins K6 and K16 were strongly expressed in lesional epidermis, suggesting that basal cell keratin expression was not down-regulated as in normal epidermis and that lesional keratinocytes mature via an abnormal pathway. When removed from the tissue and grown in culture, keratinocytes from lesional and non-lesional biopsies had similar phase microscopic morphology as well as keratin and profilaggrin expression, in contrast to the extreme differences in vivo. Lesional keratinocytes also had similar contents of keratin filaments and keratohyalin, but showed abnormal accumulation of intercellular vesicles and debris and altered cell-cell and cell-substratum interaction. Comparison of the results in tissue and in culture suggests that systemic or dermal factors influence the abnormal structural protein expression and ichthyosiform epidermal differentiation seen in CHILD syndrome, but that lesional keratinocytes maintain abnormalities in the secretion and accumulation of extracellular material in vitro similar to the lesional tissue in vivo.     

Generation and characterization of epidermolysis bullosa simplex cell lines: scratch assays show faster migration with disruptive keratin mutations

BACKGROUND: Epidermolysis bullosa simplex (EBS) is an inherited skin fragility disorder caused by mutations in keratin intermediate filament proteins. While discoveries of these mutations have increased understanding of the role of keratins and other intermediate filaments in epithelial tissues, progress towards the development of therapy for these disorders is much slower. OBJECTIVES: Cell culture model systems that display these structural defects are needed for analysis of the cellular consequences of the mutations and to enable possible therapeutic strategies to be developed. Our aim was to generate immortalized cell lines as such model systems for the study of EBS. METHODS: We generated a series of stable cell lines expressing EBS-associated keratin mutations, by immortalizing keratinocytes from EBS-affected skin biopsies with either simian virus 40 (SV40) T antigen or human papillomavirus 16 (HPV16) E6/E7, and assessed their keratin expression (by immunofluorescence), proliferation rates and migratory behaviour (in outgrowth and scratch wound assays). RESULTS: Clonal immortalized keratinocyte cell lines KEB-1, KEB-2, KEB-3 (using SV40 T antigen) and KEB-4, KEB-7 and NEB-1 (using HPV16 E6/E7) were established. These include two lines from a single individual with Weber-Cockayne EBS (i.e. KEB-3 and KEB-4, mutation K14 V270M), and three cell lines from a second family, two from siblings carrying the same mutation (KEB-1, KEB-2 lines from Dowling-Meara EBS, mutation K5 E475G) and one from an unaffected relative (NEB-1). The sixth cell line (KEB-7), with a previously unreported severe mutation (K14 R125P), was the only one to show keratin aggregates in resting conditions. Despite variations in the immortalization procedure, there was no significant difference between cell lines in keratin expression, outgrowth capabilities or response to transient heat shock. However, cell migration, as measured by speed of scratch wound closure, was significantly faster in cells with severe EBS mutations. CONCLUSIONS: These cell lines provide useful culture systems in which to assess aspects of EBS-induced cell changes. The faster migration after scratch wounding of the EBS keratinocytes may be a consequence of the known upregulation of stress-activated kinase pathways in these cells.     

角蛋白K14反义寡核苷酸对角质形成细胞增殖的影响

目的：研究角蛋白K14反义寡核苷酸(ASODN)对人角质形成细胞(KC)体外增殖活性的影响。方法：利用脂质体将人工合成的正义、反义及错配K14寡核苷酸基因片段导人体外培养的角质形成细胞，应用细胞生长抑制实验、透射电镜(TEM)和流式细胞仪(FCM)检测ASODN对角质形成细胞的增生、超微结构改变和细胞增殖周期的影响。结果：脂质体介导的K14反义寡核苷酸组KC增殖活性受到明显抑制；电镜下可见KC增殖活性受到抑制的改变，角蛋白合成明显减少；流式细胞仪检测见细胞周期发生明显改变，G1期细胞百分率上升，S期细胞百分率下降。而正义寡核苷酸组、错义寡核苷酸组及空白对照组均无此改变。结论：K14反义寡核苷酸可抑制KC体外增殖活性，应用反义寡核苷酸技术封闭K14基因，有望为银屑病的基因治疗提供新的思路。     

靶向角蛋白17基因的小干涉RNA对角质形成细胞增生与凋亡的影响

目的利用RNA干涉技术诱导角蛋白17(K17)基因沉默,观察其对角质形成细胞(KC)增生和凋亡等生物学活性的影响。方法合成两条含有针对人K17mRNA序列的正义和反义寡核苷酸,退火后与表达载体psilencer3.1-H1neo相连接,经鉴定后转染人角质形成细胞系HaCaT,分别以逆转录聚合酶链反应(RT-PCR)和免疫印迹法(W est-ern b lot)检测转染细胞K17 mRNA与蛋白水平的改变,用流式细胞仪检测转染细胞的细胞周期及凋亡情况,并通过透射电镜观察细胞的凋亡。结果成功构建了靶向人K17基因的siRNA表达载体psilencer3.1/K17,检测到瞬时转染的HaCaT细胞中K17的蛋白水平及mRNA水平均明显下降。流式细胞仪检测表明转染细胞的细胞周期发生了明显的G1期阻滞并证实凋亡的存在,电镜下观察到凋亡小体。结论对于增生活跃的角质形成细胞,K17的表达对其增生、分化和凋亡等生物学活性具有重要影响。靶向K17的siRNA能够抑制角质形成细胞增生,诱导其凋亡。     

Cadherin-mediated cell-cell contact regulates keratinocyte differentiation

Cell-extracellular matrix (ECM) and cell-cell interactions regulate keratinocyte cell fate and differentiation. In the present analysis, we examined the differentiation of primary human keratinocytes cultured on micropatterned substrates that varied the extent of cell-cell contact while maintaining constant cell-ECM areas. Bowtie-shaped micropatterned areas (75-1600 microm(2)) were engineered to either permit or prevent cell-cell contact for pairs of adherent keratinocytes. Cell pairs with direct cell-cell contact exhibited enhanced expression of the differentiation markers involucrin and keratin 10 compared to cells with no cell-cell contact. In contrast, available cell-spreading area, as regulated by pattern size, did not alter keratinocyte involucrin expression. Disruption of E-cadherin binding by either antibody blocking or expression of a dominant-negative receptor diminished the ability of micropattern-regulated cell-cell contact to modulate involucrin expression. These results demonstrate that cadherin-mediated cell-cell contact regulates early keratinocyte differentiation independently from changes in cell shape.     

Spontaneous keratinocyte cell lines representing early and advanced stages of malignant transformation of the epidermis

A unique series of epidermal cell lines representing different stages of malignant transformation were spontaneously derived from a single adult immunosuppressed individual. Four keratinocyte lines (PM1-4) established from forehead skin are here compared with 4 squamous cell carcinoma (SCC) lines (MET1-4) derived respectively from a primary cutaneous tumour, two local recurrences and a distant metastasis of invasive SCC. Despite altered growth properties, the PM lines retained many features of normal keratinocytes including keratin phenotype, differentiation capacity and non-tumorigenicity in athymic mice. In contrast, from early passage, the MET lines displayed markedly reduced growth requirements, abnormal differentiation, aberrant K18 expression and tumorigenicity in athymic mice. The abnormal keratin profile of individual MET lines closely reflected the keratin phenotype of the tumour of origin. Although unusual HPV types were identified in the original tissue, there was no evidence of persistent virus within any cell line and it appears that HPV is not critical for maintenance of the immortal phenotype. The PM lines were distinctly different from invasive SCC lines and are likely to be useful for studies of mutations important early in neoplastic progression. The SCC series represent primary, recurrent and metastatic carcinoma. Availability of such a series from the same individual will facilitate genetic analysis of the malignant process.     

Cholesterol depletion upregulates involucrin expression in epidermal keratinocytes through activation of p38

Cholesterol has been recently suggested to regulate the early steps of keratinocyte differentiation through lipid rafts. In many cell types, depletion of cholesterol activates signaling proteins like epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), or extracellular signal-regulated kinase (ERK) known to affect cell differentiation. In this study, we explored the effects of cholesterol depletion on the phenotype of cultured keratinocytes, using a treatment with methyl-beta-cyclodextrin (MbetaCD) to extract cholesterol and a treatment with lovastatin to inhibit cholesterol neosynthesis. Analysis of the expression of differentiation marker genes in early differentiating confluent cultures reveals that cholesterol depletion induces downregulation of keratin 14 (K14) and keratin 10 (K10) and upregulation of involucrin. MbetaCD treatment induces phosphorylation of EGFR, HER2, and ERK, but not HER3. Inhibition of EGFR with PD153035 impairs the MbetaCD-induced phosphorylation of EGFR, HER2, and ERK, but does not impair the alteration of K14, K10, or involucrin gene expression, indicating that other signaling proteins regulate this phenomenon. p38 has been suggested to regulate the expression of involucrin during keratinocyte differentiation. We found that MbetaCD treatment induces a prolonged phosphorylation of p38 in general and p38alpha in particular. An inhibition of p38 with PD169316 impairs the upregulation of involucrin mRNAs by a treatment with MbetaCD, but not by a p38delta-activating TPA treatment, which might suggest that cholesterol depletion alters involucrin gene expression through activation of p38alpha/beta.     

Changes of epidermal cell morphology and keratin expression induced by inhibitors of protein kinase C.

Several lines of evidence show protein kinase C as being involved in various regulatory processes in keratinocyte biology, e.g. proliferation and differentiation. In the present study, we investigated the effects of three different inhibitors of protein kinase C, staurosporine, CP 46'665-1, and tiflucarbine, on cell morphology and keratin expression in a non-tumorigenic human keratinocyte cell line (HaCaT cells). Staurosporine, being the most potent inhibitor of protein kinase C activity in vitro, and CP 46'665-1 induced morphological transformation to a fibroblast-like cell shape. In contrast, no changes in cell morphology were observed after exposure to tiflucarbine. The investigation of keratin expression in HaCaT cells grown in the presence of the different compounds revealed the following changes: After 72 h of cultivation, keratins 8 and 18 were still expressed in treated cells, whereas expression of keratin 13 was decreased as compared to control cells. Immunoblotting to detect vimentin demonstrated its absence in treated and control cells. Since tiflucarbine is known as a dual protein kinase C/calmodulin inhibitor whereas staurosporine and CP 46'665-1 do not antagonize calmodulin function, it might be possible that not only protein kinase C but also calmodulin is involved in the process leading to the morphological changes.     

Loss of integrin alpha9beta1 results in defects in proliferation, causing poor re-epithelialization during cutaneous wound healing

The wound microenvironment comprises constituents, such as the extracellular matrix (ECM), that regulate with temporal and spatial precision, the migratory, proliferative, and contractility of wound cells. Prompt closure of the wound is an early and critical phase of healing and beta1 integrins are important in this process. We previously reported a marked increase in integrin alpha9beta1 expression in epidermal keratinocytes in cutaneous and corneal wounds. However, the functional role of keratinocyte alpha9beta1 during re-epithelialization is unknown and analysis has been precluded by the lethal phenotype of integrin alpha9beta1 knockout mice. We now report that in conditional integrin alpha9 knockout (K14-alpha9 null) mice, normal proliferation occurs in epidermal keratinocytes and corneal basal cells. Normal epidermal keratinocyte morphology is also retained. However, corneal basal cell morphology and epithelial thickness are altered, suggesting that loss of integrin alpha9beta1 results in abnormal corneal differentiation. In cutaneous wounds, the number of proliferating epidermal keratinocytes is significantly reduced in K14-alpha9 null mice compared with alpha9(fl/-) mice, but not in Cre (control) mice. The decreased keratinocyte proliferation observed in K14-alpha9 null mice negatively impacts healing, resulting in a thinner migrating epithelium, demonstrating that alpha9beta1 is crucial for efficient and proper re-epithelialization during cutaneous wound healing.     

Numerous keratinocyte subtypes involved in wound re-epithelialization

The expression of different keratin intermediate filaments has been used to define keratinocyte maturation and different phenotypic subtypes involved in acute wound (AW) healing. Immunohistochemistry with specific anti-keratin monoclonal and polyclonal antibodies was used to examine AW in normal healthy volunteers (n = 16). In all wounds examined, basal keratinocytes and cells at the leading edge of the wound expressed keratins K5 and K14. However, suprabasal cells had a more complex pattern of keratin expression, which was dependent on their position relative to the wound and location within the suprabasal compartment of the epidermis. In general, K10 was expressed in suprabasal keratinocytes at the wound edge, but not in keratinocytes covering the wound center, which expressed K6, K16, and K17 in a complex fashion. Ki67 expression, a marker of cell proliferation, was restricted to basal and immediate suprabasal layers at the wound edge. Keratinocytes populated the wound bed below the scab by migration, which was supported by keratinocyte proliferation in the surrounding epidermis both at and adjacent to the wound edge.     

Keratin 14-null cells as a model to test the efficacy of gene therapy approaches in epithelial cells

Skin fragility disorders caused by keratin mutations are incurable, and a better understanding of their etiology is needed to find new ways to improve and treat these conditions. The best-studied skin fragility disorder is epidermolysis bullosa simplex (EBS), an autosomal dominant condition caused by mutations in keratin 5 (K5) or K14. To analyze disease mechanisms and develop gene therapy strategies, we have used keratinocyte cell lines derived from EBS patients as model systems. Here, we describe two cell lines established from EBS patients with K14-null mutations. We analyze the responses of these cells to stress assays previously shown to discriminate between wild-type and keratin-mutant keratinocytes, to directly evaluate the efficacy of rescuing K14-null cells by supplementation with wild-type K14 complementary DNA (cDNA). The K14-null cells show elevated levels of stress correlating with reduced normal keratin function. By transfecting wild-type K14 into these cells, we demonstrate "proof of principle" that an add-back approach can significantly rescue the normal keratinocyte behavior profile. These K14-null cell lines provide a disease model for studying the effects of keratin ablation in EBS patients and to test the efficacy of gene add-back and other therapy approaches in keratinocytes.