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.     

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.     

Overexpression of connexin26 in the basal keratinocytes reduces sensitivity to tumor promoter TPA

Please cite this paper as: Overexpression of connexin26 in the basal keratinocytes reduces sensitivity to tumor promoter TPA. Experimental Dermatology 2010; 19: 633–640. Abstract: Connexin 26 is important in keratinocyte proliferation, differentiation and skin pathologies. Cx26 is barely expressed in normal adult epidermis, but its expression is induced during wound healing, psoriasis, and skin hyperplasia stimulated by tumor promoters. In hyperplastic proliferating epidermis, Cx26 is co‐expressed with Cx43 typical for basal and suprabasal keratinocytes. As Cx26 and Cx43 can not form permeable gap junctions, their co‐expression may alter the gap junctional communication between keratinocytes and induce proliferation. To test the effect of persistent co‐expression of Cx26 and Cx43 in epidermis, we generated transgenic mice using keratin5 promoter to target Cx26 to basal Cx43‐positive keratinocytes. We evaluated the effect of ectopic Cx26 on keratinocyte proliferation and differentiation in normal and 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA)‐treated skin. The ectopic Cx26 expression in epidermis did not significantly affect skin development, keratinocyte differentiation and proliferation in newborn and adult skin. Unexpectedly, the proliferative effect of tumor promoter TPA was strongly decreased in epidermis of K5.Cx26 transgenics. This correlated with significant down‐regulation of TPA‐induced activity of protein kinase C (PKC) in K5.Cx26 mice.     

Fibroblast growth factor 10 induces proliferation and differentiation of human primary cultured keratinocytes

Fibroblast growth factor 10 is a novel member of the fibroblast growth factor family, which is involved in morphogenesis and epithelial proliferation. It is highly homologous to the keratinocyte growth factor (or fibroblast growth factor 7), a key mediator of keratinocyte growth and differentiation. Both fibroblast growth factor 10 and keratinocyte growth factor bind with high affinity to the tyrosine kinase keratinocyte growth factor receptor. Here we analyzed the effect of fibroblast growth factor 10 on primary cultures of human keratinocytes, grown in chemically defined medium, and we compared the proliferative and differentiative cell responses to fibroblast growth factor 10 with those induced by keratinocyte growth factor and epidermal growth factor. Cell counting, 5-bromo-2'-deoxyuridine incorporation, and western blot analysis showed that fibroblast growth factor 10, similarly to keratinocyte growth factor, not only is a potent mitogen for human keratinocytes, but also promotes the expression of both early differentiation markers K1 and K10 and late differentiation marker filaggrin in response to the Ca2+ signal, and seems to sustain the proliferative activity in suprabasal stratified cells. Immunoprecipitation/western blot analysis revealed that fibroblast growth factor 10, similarly to keratinocyte growth factor, is able to induce tyrosine phosphorylation of keratinocyte growth factor receptor and of cellular substrates such as PLCgamma.     

The expression of keratinocyte growth factor receptor (FGFR2-IIIb) correlates with the high proliferative rate of HaCaT keratinocytes

Keratinocyte growth factor receptor (KGFR = FGFR2-IIIb) is a tyrosine kinase receptor expressed by keratinocytes, which mediates the effects of fibroblast growth factors (FGF). There are contradictory data in the literature regarding the role of FGFR2-IIIb during the proliferation/differentiation programme of keratinocytes. In this study, we aimed to investigate whether overexpression of FGFR2-IIIb may have a role in the regulation of keratinocyte proliferation. We analysed the expression of FGFR2-IIIb in an in vitro HaCaT model system representing different stages of proliferation and differentiation of keratinocytes. Real-time RT-PCR and Western blot analyses demonstrated a correlation between FGFR2-IIIb mRNA and protein expression and the proportion of cells in S/G2/M phase in synchronized HaCaT keratinocytes and thus with proliferation activity (r = 0.96). After treatment with the antipsoriatic drug, dithranol, FGFR2-IIIb is downregulated dose dependently both at mRNA and protein levels. Moreover, when the rate of proliferation is decreased by the lack of cell attachment to the culturing surface, FGFR2-IIIb mRNA (P = 0.0315) and protein expressions were also reduced (P = 0.0242), while a differentiation marker, keratin 10, mRNA (P = 0.0003) and protein levels (P = 0.001) were increased (r = -0.92). Based on our results we conclude that FGFR2-IIIb expression in HaCaT keratinocytes corresponds with the proliferative activation of the cells and is not related to the differentiation programme.     

The expression of differentiation markers in aquaporin-3 deficient epidermis

Aquaporin-3 (AQP3) is a water/glycerol transporting protein expressed strongly at the plasma membrane of keratinocytes. There is evidence for involvement of AQP3-facilitated water and glycerol transport in keratinocyte migration and proliferation, respectively. Here, we investigated the involvement of AQP3 in keratinocyte differentiation. Studies were done using AQP3 knockout mice, primary cultures of mouse keratinocytes (AQP3 knockout), neonatal human keratinocytes (AQP3 knockdown), and human skin. Cells were cultured with high Ca²⁺ or 1α,25-dihydroxyvitamin D₃ (VD₃) to induce differentiation. The expression of differentiation marker proteins and differentiating responses were comparable in control and AQP3-knockout or knockdown keratinocytes. Topical application of all-trans retinoic acid (RA), a known regulator of keratinocyte differentiation and proliferation, induced comparable expression of differentiation marker proteins in wildtype and AQP3 null epidermis, though with impaired RA-induced proliferation in AQP3 null mice. Immunostaining of human and mouse epidermis showed greater AQP3 expression in cells undergoing proliferation than differentiation. Our results showed little influence of AQP3 on keratinocyte differentiation, and provide further support for the proposed involvement of AQP3-facilitated cell proliferation.     

银屑病皮损内酪氨酸蛋白激酶活性的初步研究

目的 探讨酪氨酸蛋白激酶(TPK)在银屑病表皮异常增生中的作用。方法 通过外源性底物磷酸化的方法,测定银屑病进行期皮损角质形成细胞的TPK活性。结果 ①银屑病进行期皮损角质形成细胞基础TPK活性显著增高,且对于转化生长因子-α的刺激,反应性明显增强;②增高的基础TPK活性与角质形成细胞膜表皮生长因子受体的表达量呈明显正相关;同时银屑病角质形成细胞表皮生长因子受体的自磷酸化水平亦显著增高。结论 TPK活性增高,以及对有丝分裂信号刺激的反应增强,可能在表皮的增生中起到重要作用,TPK活性的改变可能主要是由于表皮生长因子受体的表达异常所致,表皮生长因子受体可能在银屑病表皮的异常增生中发挥着重要作用。     

皮肤病中蛋白激酶D1研究进展

蛋白激酶D1是一种在体内多个重要器官广泛表达的钙离子/钙调蛋白依赖性的丝氨酸/苏氨酸蛋白激酶,参与多种重要的生理和病理活动,在许多肿瘤组织中表达异常。PKD1促进角质形成细胞增殖,抑制其分化,对表皮伤口愈合及肿瘤的形成具有促进作用,与UVB、ROS等影响皮肤疾病发生发展的重要因素之间都有密切联系。因此PKD1可能成为治疗某些皮肤病甚至皮肤肿瘤更有效的分子靶点。     

Insulin-like growth factor binding protein-3 (IGFBP-3) localizes to and modulates proliferative epidermal keratinocytes in vivo

BACKGROUND: The colocalization of insulin-like growth factor binding protein-3 (IGFBP-3) and IGF-I receptor (IGF-IR) in the basal/germinative layer of the epidermis suggests a key role in modulating epidermal homeostasis. OBJECTIVES: We aimed to clarify both the specific cellular localization and the effect of excess epidermal IGFBP-3 on keratinocyte proliferation. METHODS: (i) Total RNA was isolated from fluorescence-activated cell sorted basal human keratinocyte subtypes [keratinocyte stem cells, transit amplifying keratinocytes (TA), postmitotic differentiating keratinocytes (PMD)], and real-time polymerase chain reaction analysis was used to determine the abundance of IGFBP-3 and IGF-IR mRNAs. (ii) An IGFBP-3 transgenic mouse model was then used to assess the effect of excess epidermal IGFBP-3 on keratinocyte proliferation. Excess epidermal IGFBP-3 mRNA and protein was determined by in situ hybridization and immunohistochemistry, respectively. RESULTS: (i) The highest levels of IGFBP-3 mRNA were detected in TA keratinocytes, in contrast to IGF-IR mRNA levels which were highest in PMD keratinocytes. (ii) Elevated human IGFBP-3 mRNA and protein was confirmed in the epidermis of skin derived from transgenic mice. Excess IGFBP-3 reduced the relative percentage of proliferative keratinocytes (Ki67 positive) irrespective of skin location (belly, back and tail). Thus, in the epidermis, IGFBP-3 mRNA is highly expressed by proliferative keratinocytes (TA) and overexpression of IGFBP-3 inhibits keratinocyte proliferation. CONCLUSIONS: We conclude that in vivo IGFBP-3 ensures epidermal homeostasis via downregulation of keratinocyte proliferation, and thus modulates the early stages of keratinocyte differentiation.     

Regulated synthesis of low-molecular-weight antigens in keratinocyte cell cultures

Proteins from mouse epidermis cytosol extracts react on immunoblots with a polyclonal rabbit antiserum raised against rat skin calcium-binding protein (SCaBP), a parvalbumin of the panniculus carnosus. Three mouse epidermal proteins with molecular weights between 10-12K, which are distinct from SCaBP, are recognized by the antiserum. The synthesis of these proteins in keratinocyte culture is modulated by Ca++, as is the differentiation of the keratinocytes. Proliferating mouse keratinocytes in medium containing 0.07 mM Ca++ (low Ca++) undergo terminal differentiation when the Ca++ concentration is elevated to 1.8 mM (high Ca++). Synthesis of the 3 antigens can be demonstrated when soluble extracts of keratinocytes labeled with [35S]methionine in low Ca++ medium are immunoprecipitated with anti-SCaBP serum. These antigens are not synthesized in cultures of dermal fibroblasts. When keratinocytes are switched to high Ca++ medium, synthesis of these antigens is greatly diminished over the course of 48-72 h. However, the antigens persist in differentiating cells. When proliferating keratinocytes in low Ca++ medium are exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), differentiation is induced in a subpopulation of cells, and specific antigen synthesis is transiently inhibited. The inhibition correlates with the time when many cells are differentiating in response to TPA. When proliferating keratinocytes are pulse-labeled with 32PO4, the 11K antigen is phosphorylated and the phosphorylation is not enhanced by TPA exposure. All 3 antigens are synthesized in a reticulocyte lysate preparation with added newborn mouse epidermis messenger RNA or mRNA from keratinocytes cultured in low Ca++ medium. Thus, these antigens are likely to represent unique proteins rather than processed or degraded ones. The coordinately regulated expression of these antigens associated with the differentiation state of the keratinocytes suggests that these proteins are important in keratinocyte proliferation and differentiation.     

Rosiglitazone inhibits proliferation, motility, and matrix metalloproteinase production in keratinocytes

This study was undertaken to evaluate the effects of thiazolidinediones (TZD) on keratinocyte proliferation, motility, and matrix metalloproteinase (MMP) production. Rosiglitazone (a potent TZD) inhibited both proliferation and motility as well as elaboration of MMP-1 and MMP-9. Inhibition was obtained with keratinocytes in monolayer culture and human skin in organ culture. There were significant concentration-response differences in sensitivity of the three keratinocyte responses to treatment with rosiglitazone. In contrast to keratinocytes, dermal fibroblasts were resistant to the effects of rosiglitazone. Treatment of keratinocytes with rosiglitazone did not suppress epidermal growth factor receptor autophosphorylation, but inhibited signaling through the extracellular regulated kinase mitogen-activated protein kinase pathway without a concomitant effect on pathways that lead to c-jun activation. Pioglitazone, another TZD, also suppressed keratinocyte proliferation, although it was less effective than rosiglitazone. An experimental TZD (BP-1107) inhibited keratinocyte proliferation at a much lower concentration than either rosiglitazone or pioglitazone. Because enhanced keratinocyte motility and increased MMP production as well as increased keratinocyte proliferation are thought to contribute to the phenotype of psoriatic lesional skin, we propose that interference with these keratinocyte responses contributes to the previously reported antipsoriatic activity of TZD.     

Enhancement of Keratinocyte Differentiation by Rose Absolute Oil

Background

Through differentiation processes, keratinocytes provide a physical barrier to our bodies and control skin features such as moisturization, wrinkles and pigmentation. Keratinocyte differentiation is disturbed in several skin diseases such as psoriasis and atopic dermatitis.

Objective

The aim of this study is to evaluate the keratinocyte differentiation-enhancing effect of rose absolute oil (RAO).

Methods

Primary cultured human normal keratinocytes were treated with RAO, and differentiation then checked by the expression of marker genes.

Results

RAO did not induce cytotoxicity on cultured keratinocytes at a dose of 10µM. The level of involucrin, an early marker for keratinocyte differentiation, was significantly increased by RAO. Concomitantly, RAO increased involucrin promoter activity, indicating that RAO increased involucrin gene expression at the mRNA level. Furthermore, RAO increased the level of filaggrin in cultured keratinocytes, and in the granular layer of mouse skin. In line with these results, RAO decreased the proliferation of keratinocytes cultured in vitro. When RAO was applied topically on the tape-stripped mouse skins, it accelerated the recovery of disturbed barrier function.

Conclusion

These results suggest that RAO may be applicable for the control of skin texture and keratinocyte differentiation-related skin diseases.     

Role of Keratinocytes in the Development of Vitiligo

Vitiligo is an acquired depigmentary disorder of the skin that results from the loss of functioning epidermal melanocytes. Most studies on vitiligo have concentrated on the abnormality of melanocytes rather than the abnormality of keratinocytes; however, epidermal melanocytes form a functional and structural unit with neighboring keratinocytes. In fact, direct cell-to cell contact stimulates in vitro proliferation of melanocytes, and growth factors produced by adjacent keratinocytes regulate the proliferation and differentiation of melanocytes. The potential role of keratinocyte-derived cytokines has also been presented. We focused on the structural changes in vitiliginous keratinocytes, which may result in loss of melanocytes, to examine the pathomechanism of vitiligo. The results of a comparison between depigmented and normally pigmented epidermis in patients with vitiligo showed that the keratinocytes in the depigmented epidermis were more vulnerable to apoptosis. Impaired Phosphatidylinositol 3-kinase (PI3K)/serine/threonine protein kinase (Akt) activation followed by reduced nuclear factor-κB activation under increased tumor necrosis factor-α levels was demonstrated as a mechanism for keratinocyte apoptosis. The role of aquaporin 3 in keratinocyte apoptosis was addressed based on the relationship between the PI3K/AKT pathway and the E-cadherin-catenin complex. Apoptotic keratinocytes induced a lower expression of keratinocyte-derived factors, including stem cell factor, in depigmented epidermis, resulting in passive melanocyte death.     

Activation of kinin B receptor triggers differentiation of cultured human keratinocytes

Background Keratinocyte life span is modulated by receptors that control proliferation and differentiation, key processes during cutaneous tissue repair. The kinin B₁ receptor (B₁R) has been reported in normal and pathological human skin, but so far there is no information about its role in keratinocyte biology. Objectives To determine the consequence of kinin B₁R stimulation on tyrosine phosphorylation, a key signalling mechanism involved in keratinocyte proliferation and differentiation. Methods Subconfluent primary cultures of human keratinocytes were used to investigate tyrosine phosphorylation, epidermal growth factor receptor (EGFR) transactivation, cell proliferation and keratinocyte differentiation. Cell proliferation was assessed by measuring bromodeoxyuridine incorporation whereas assessment of cell differentiation was based on the expression of filaggrin, cytokeratin 10 (CK10) and involucrin. Results The major proteins phosphorylated, after B₁R stimulation, were of molecular mass 170, 125, 89 and 70 kDa. The 170‐ and 125‐kDa proteins were identified as EGFR and p125^FAK, respectively. Phosphorylation was greatly reduced by GF109203X and by overexposure of keratinocytes to phorbol 12‐myristate 13‐acetate, indicating the participation of protein kinase C. B₁R stimulation did not increase [Ca²⁺]i, but triggered EGFR transactivation, an event that involved phosphorylation of Tyr⁸⁴⁵, Tyr⁹⁹² and Tyr¹⁰⁶⁸ of EGFR. B₁R stimulation did not elicit keratinocyte proliferation, but triggered cell differentiation, visualized as an increase of filaggrin, CK10 and involucrin. Blockade of EGFR tyrosine kinase by AG1478, before B₁R stimulation, produced an additional increase in filaggrin expression. Conclusions The kinin B₁R may contribute to keratinocyte differentiation and migration by triggering specific tyrosine signalling pathways or by interacting with the ErbB receptor family.     

Interferon regulatory factor 6 is necessary, but not sufficient, for keratinocyte differentiation

Regulation of epidermal proliferation and differentiation is critical for maintenance of cutaneous homeostasis. Interferon Regulatory Factor 6 (Irf6)-deficient mice die perinatally and exhibit ectopic proliferation and defective epidermal differentiation. We sought to determine whether these disruptions of epidermal function were cell autonomous, and used embryonic Irf6(-/-) keratinocytes to understand the specific role of Irf6 in keratinocyte proliferation and differentiation. In the absence of Irf6, keratinocytes exhibited a heterogeneous phenotype with the presence of large cells. Irf6(-/-) keratinocytes displayed increased colony-forming efficiency compared with wild-type cells, suggesting that Irf6 represses long-term proliferation. Irf6 was present at low levels in wild-type keratinocytes in culture, and upregulated after induction of differentiation in vitro, along with upregulation of markers of early differentiation. However, Irf6(-/-) keratinocytes did not express markers of terminal differentiation. Overexpression of Irf6 in wild-type keratinocytes was insufficient to induce expression of markers of differentiation under growing conditions. Together, these results indicated that Irf6 is necessary, but not sufficient, for keratinocyte differentiation. Finally, using a transgenic mouse expressing Lac-Z under the regulation of an enhancer element 9.7 kb upstream of the Irf6 start site, we demonstrated that this element contributes to the regulation of Irf6 in the epidermis and keratinocytes in culture.     

Interferon gamma-induced PNA-binding glycoproteins as markers of human keratinocyte differentiation: biological evidence using protein kinase C agonists, antagonists and retinoic acid

Membrane glycoproteins (gps) play an important role in cell-cell interactions during epidermal maturation, and we have previously shown an up-regulation of PNA-binding gps in cultured human keratinocytes treated with interferon gamma (IFN-γ). The protein kinase C (PKC) pathway is known to play a key role in the regulation of proliferation and differentiation of keratinocytes and is also reported to be involved in some IFN-γ-mediated effects. In order to evaluate the cellular mechanisms and whether PNA-binding gp expression is related to the differentiative activity of the lymphokine, we studied the effects of PKC agonists and antagonists and the role of retinoic acid (RA), in the induction of these gps in cultured human keratinocytes stimulated with IFN-γ and processed for protein analysis. The expression of PNA-binding gps was revealed by incubation of SDS-polyacrylamide gels with ¹²⁵ I-PNA. The PKC antagonists (H7, sphingosine) as well as RA downgregulated the IFN-γ-induced PNA-reactive gps, whereas staurosporine and TPA upregulated their expression. These results provide evidence that PNA-reactive gps are late highly IFN-γ-sensitive markers of keratinocyte differentiation, drastically modulated through selective isoforms of PKC. Received: 3 March 1997