| 1. |
- Belsõ, Nóra, et al.
(författare)
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Differential expression of D-type cyclins in HaCaT keratinocytes and in psoriasis.
- 2008
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Ingår i: Journal of Investigative Dermatology. - : Elsevier BV. - 0022-202X .- 1523-1747. ; 128:3, s. 634-42
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Tidskriftsartikel (refereegranskat)abstract
- In this study, we show that the G0-G1/S phase of HaCaT keratinocyte cell cycle is characterized by D1-type cyclin expression, whereas during the repeated rapid turnover of highly proliferating cells, the expression of cyclins D2 and D3 dominates. Knocking down cyclin D1 mRNA resulted in no change of cell proliferation and morphology, indicating that D2 and D3 cyclins could substitute for D1 in driving the cell cycle. Increased numbers of cyclin D1-expressing keratinocytes were found in the basal layers of the lesional psoriatic epidermis compared to both normal and non-lesional epidermis without increased expression of cyclin D1 mRNA, suggesting a possible dysfunction in the degradation of cyclin D1 protein. We also detected a significant increase in cyclin D2 and D3 mRNA expressions in psoriatic epidermis compared to normal epidermis with no difference in protein expressions. Blocking alpha5-integrin function by a neutralizing antibody in HaCaT keratinocytes downregulated the expression of cyclin D1 mRNA without affecting the expressions of cyclin D2 and D3 indicating a regulatory role for alpha5-integrin in the expression of cyclin D1. Our data suggest a possible role for D-type cyclins in the excessive basal-cell proliferation and perturbed keratinocyte differentiation in the psoriatic epidermis.
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| 2. |
- Nagy, Nikoletta, et al.
(författare)
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The expression of keratinocyte growth factor receptor (FGFR2-IIIb) correlates with the high proliferative rate of HaCaT keratinocytes.
- 2006
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Ingår i: Experimental dermatology. - : Wiley. - 0906-6705 .- 1600-0625. ; 15:8, s. 596-605
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Pivarcsi, Andor, et al.
(författare)
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Differentiation-regulated expression of Toll-like receptors 2 and 4 in HaCaT keratinocytes.
- 2004
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Ingår i: Archives of Dermatological Research. - : Springer Science and Business Media LLC. - 0340-3696 .- 1432-069X. ; 296:3, s. 120-4
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Tidskriftsartikel (refereegranskat)abstract
- Toll-like receptors (TLRs) play an important role in the recognition of pathogens in keratinocytes. In this study, we investigated whether the differentiation state of HaCaT keratinocytes correlates with the expression of TLR2 and TLR4 genes. The expression levels of TLR2 and TLR4 in a HaCaT differentiation model system were determined using quantitative real-time RT-PCR (Q-RT-PCR) and flow cytometry. The progression of keratinocyte differentiation was monitored by determining the level of involucrin gene expression using Q-RT-PCR. The expression levels of TLR2 and TLR4 increased with the stage of differentiation and there were strong correlations between the expression level of the involucrin gene and those of the TLR2 gene ( r=0.809, P<0.0001) and the TLR4 gene ( r=0.568, P<0.02). Increased cell surface expression of TLR2 and TLR4 was also found in differentiated HaCaT keratinocytes by flow cytometric analysis. Our findings suggest that upregulation of TLR expression during differentiation in keratinocytes could be a part of the differentiation process of keratinocytes and could have biological significance in protecting skin against microbes.
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| 4. |
- Szabad, Gábor, et al.
(författare)
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Human adult epidermal melanocytes cultured without chemical mitogens express the EGF receptor and respond to EGF.
- 2007
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Ingår i: Archives of Dermatological Research. - : Springer Science and Business Media LLC. - 0340-3696 .- 1432-069X. ; 299:4, s. 191-200
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Tidskriftsartikel (refereegranskat)abstract
- We describe a novel chemical mitogen-free in vitro culture technique for obtaining pure melanocyte cultures using normal human adult epidermis as a source. The culture medium consists equal parts of the commercially available Keratinocyte Basal and AIM-V media (both from Gibco), as basal medium, which is supplemented with fetal bovine serum, bovine pituitary extract and recombinant human epidermal growth factor (EGF). Melanocytes harvested from human adult skin proliferate extensively and can be passaged serially up to 10-15 times using this medium. We have verified the identity of the cultured cells by tyrosinase mRNA expression and TRP-1 protein staining. Moreover, we showed that autologous human serum alone, without additional supplements is able to provide sufficient growth support for the cultured cells in the basal medium, making this culture technique suitable for autologous melanocyte transplantation. In this culture system normal human adult melanocytes expressed both EGF receptor (EGFR) mRNA and protein and EGF showed a dose dependent mitogenic effect on the cells. EGF itself had no significant influence on EGFR mRNA expression.
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| 5. |
- Széll, Márta, et al.
(författare)
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Proliferating keratinocytes are putative sources of the psoriasis susceptibility-related EDA+ (extra domain A of fibronectin) oncofetal fibronectin.
- 2004
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Ingår i: Journal of Investigative Dermatology. - 0022-202X .- 1523-1747. ; 123:3, s. 537-46
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Tidskriftsartikel (refereegranskat)abstract
- The extra domain A of fibronectin (EDA+ oncofetal isoform of fibronectin was recently reported to be overexpressed in psoriatic uninvolved epidermis. It has been proposed that the abnormal presence of EDA+ oncofetal protein at the dermal-epidermal junction in the uninvolved skin may provide the "psoriatic" environment in which keratinocytes are in a preactivated state with regard to mitogenic signals (e.g., T cell lymphokines). To determine the possible sources of cellular fibronectin in the non-lesional psoriatic skin, we aimed to investigate whether keratinocytes could produce the EDA+ oncofetal form of fibronectin. RT-PCR studies revealed that both cultured normal keratinocytes and HaCaT cells express the EDA+ splice variant of fibronectin mRNA, and in HaCaT cells the EDA+/EDA- transcript ratio was elevated compared with normal keratinocytes. Cultured keratinocytes and HaCaT cells showed intracytoplasmic staining with an EDA+ fibronectin-specific antibody and among the positively stained cells many showed mitosis. Using RT-PCR, western blot analysis, and flow cytometry, we showed that in synchronized HaCaT cells the amount of both total fibronectin and its EDA+ isoform change with the proliferation/differentiation state of HaCaT cells and peak in highly proliferating cells. We show that in short-term ex vivo cultures, a small population of EDA+ fibronectin containing cell population appear among psoriatic uninvolved, but not normal epidermal cells. We also demonstrate that cell attachment has a strong influence on the expression of both total and EDA+ fibronectin. Our results suggest that proliferating keratinocytes could be the sources of the psoriasis susceptibility-related EDA+ oncofetal fibronectin in the epidermis.
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