Differentiation Of HaCaT Cells Research Articles

The decline in cellular iron is crucial for differentiation in keratinocytes.

Iron is a vital metal for most biological functions in tissues, and its concentration is exquisitely regulated at the cellular level. During the process of differentiation, keratinocytes in the epidermis undergo a noticeable reduction in iron content. Conversely, psoriatic lesions, characterized by disruptions in epidermal differentiation, frequently reveal an excessive accumulation of iron within keratinocytes that have undergone differentiation. In this study, we clarified the significance of attenuated cellular iron content in the intricate course of epidermal differentiation. We illustrated this phenomenon through the utilization of hinokitiol, an iron chelator derived from the heartwood of Taiwanese hinoki, which forcibly delivers iron into cells independent of the intrinsic iron-regulation systems. While primary cultured keratinocytes readily succumbed to necrotic cell death by this iron chelator, mild administration of the hinokitiol-iron complex modestly disrupts the process of differentiation in these cells. Notably, keratinocyte model cells HaCaT and anaplastic skin rudiments exhibit remarkable resilience against the cytotoxic impact of hinokitiol, and the potent artificial influx of iron explains a suppressive effect selectively on epidermal differentiation. Moreover, the augmentation of iron content induced by the overexpression of divalent metal transporter 1 culminates in the inhibition of differentiation in HaCaT cells. Consequently, the diminution in cellular iron content emerges as an important determinant influencing the trajectory of keratinocyte differentiation.

Involvement of CacyBP/SIP in differentiation and the immune response of HaCaT keratinocytes

CacyBP/SIP is a multifunctional protein present in various cells and tissues. However, its expression and role in the epidermis has not been explored so far. In this work, using RT-qPCR, Western blot analysis and three-dimensional (3D) organotypic cultures of HaCaT keratinocytes we show that CacyBP/SIP is present in the epidermis. To investigate the possible role of CacyBP/SIP in keratinocytes we obtained CacyBP/SIP knockdown cells and studied the effect of CacyBP/SIP deficiency on their differentiation and response to viral infection. We found that CacyBP/SIP knockdown results in reduced expression of epidermal differentiation markers in both undifferentiated and differentiated HaCaT cells. Since epidermis is engaged in immune defense, the impact of CacyBP/SIP knockdown on this process was also analyzed. By applying RT-qPCR and Western blot it was found that poly(I:C), a synthetic analog of double-stranded RNA that mimics viral infection, stimulated the expression of genes involved in antiviral response, such as IFIT1, IFIT2 and OASL. Interestingly, following poly(I:C) stimulation, the level of expression of these genes was significantly lower in cells with CacyBP/SIP knockdown than control ones. Since the signaling pathway mediating cellular responses to viral infection involves, among others, the STAT1 transcription factor, we measured its activity using luciferase assay and found that it was lower in CacyBP/SIP knockdown HaCaT cells. Altogether, the presented results indicate that CacyBP/SIP promotes epidermal differentiation and might be involved in response of the skin cells to viral infection.

Effect of Zinc Ions on the Proliferation and Differentiation of Keratinocytes

Background: Psoriasis is a chronic inflammatory disease in which there is hyperproliferation and abnormal differentiation of keratinocytes. Since high levels of KLK7, an enzyme inhibited by zinc (Zn2+) ions, are present in psoriatic lesions, we have studied the effect of zinc ions in the viability of keratinocytes, as well as in the activity of KLK5 and KLK7 and in the expression of epidermal markers. Methods and Results: The cells were cultured in the absence or presence of Zn2+ ions (5.0, 10 and 25 M). Cell viability was evaluated by the MTT method after during 14 days. Cell death was evaluated by flow cytometry using propidium iodide. The activity of the KLK was evaluated on the hydrolysis of synthetic substrates. Expression of involucrin, filaggrin, cytokeratins (CK) 5, 10 and 14 was evaluated by quantitative PCR. Cell incubation with Zn2+ ions did not result in significant changes in cell viability. By MTT assay, it was observed that the cultures incubated with 10 and 25 M Zn2+ ions showed a decrease in the number of viable cells in comparison to the control. Cells cultured for 1 day in the presence of 25 M Zn2+ ions displayed a decrease in KLK7 activity. In the presence of Zn2+ ions, it was shown an increase in the expression of CK5, 10 and 14, involucrin and filaggrin. Conclusions: These results have shown that zinc ions can affect the differentiation of HaCat cells, contributing for future therapeutic trials related to psoriasis based on the modulation of KLK activity.

Konjac Ceramide (kCer)-Mediated Signal Transduction of the Sema3A Pathway Promotes HaCaT Keratinocyte Differentiation.

Simple SummaryKonjac ceramide (kCer) is a unique molecular species of plant-type ceramide, and is a potential Sema3A-like ligand of Nrp1. kCer suppresses histamine-stimulated cell migration of HaCaT keratinocytes. This effect of kCer is not due to histamine-activated GPCRs, but rather to Sema3A-Nrp1 receptor binding. The present study focused on the ability of kCer to induce cell differentiation, in addition to its anti-migratory effects. We demonstrated that the effects of kCer on cell migration and cell differentiation are perpetuated by a cascade of crosstalk between pathways downstream of Nrp1 and GPCR in HaCaT cells.Histamines suppress epidermal keratinocyte differentiation. Previously, we reported that konjac ceramide (kCer) suppresses histamine-stimulated cell migration of HaCaT keratinocytes. kCer specifically binds to Nrp1 and does not interact with histamine receptors. The signaling mechanism of kCer in HaCaT cells is also controlled by an intracellular signaling cascade activated by the Sema3A-Nrp1 pathway. In the present study, we demonstrated that kCer treatment induced HaCaT keratinocyte differentiation after migration of immature cells. kCer-induced HaCaT cell differentiation was accompanied by some features of keratinocyte differentiation markers. kCer induced activating phosphorylation of p38MAPK and c-Fos, which increased the protein levels of involucrin that was the latter differentiation marker. In addition, we demonstrated that the effects of both kCer and histamines are regulated by an intracellular mechanism of Rac1 activation/RhoA inhibition downstream of the Sema3A/Nrp1 receptor and histamine/GPCR pathways. In summary, the effects of kCer on cell migration and cell differentiation are regulated by cascade crosstalk between downstream Nrp1 and histamine-GPCR pathways in HaCaT cells.

Quantitative structure property relationship assisted development of Fluocinolone acetonide loaded transfersomes for targeted delivery

Fluocinolone acetonide (FA) is a corticosteroid, synthetic hydrocortisone used to reduce skin inflammation and itching in psoriasis. The present study investigate the formulation of nanotransfersomes containing FA (FA-NTs) using Quantitative Structure Property Relationship (QSPR) and Box–Behnken approach. FA-NTs containing phospholipid, edge activator and cholesterol in definite ratio were prepared by high pressure homogenization and optimized using Box–Behnken design. Based on the QSPR analysis, Phospholipon 90H was selected for nanotransfersome form along with sodium deoxycholate as edge activator. The impact of independent variables Phospholipid concentration (X1), sodium deoxycholate (X2) and number of homogenization cycles (X3) on dependent variables viz., particle size and entrapment efficiency (%EE) were studied. The optimized nanotransfersomal formulation was characterized for FTIR, XRD, DSC, particle size, zeta potential, entrapment efficiency, in-vitro release and ex-vivo permeation studies. FA-NTs batch evidenced 92 ± 4% entrapment efficiency and showed 29.9 nm particle size, 0.264 polydispersity index and −23.72 mV zeta potential. FA-NTs exhibited 68% drug release within 24 h and followed Higuchi release kinetic model (R2 = 0.9902). FA-NTs showed 22.56 μg/cm2/h transdermal flux with 12.40 fold higher than FA dispersion. Biologically, FA-NTs showed hyperproliferation inhibition of differentiated HaCaT cells as evidenced from cell viability, antiproliferative action, nitric oxide production, proinflammatory cytokine measurement, Quantitative Real time PCR and cellular uptake studies. Thus, the present study demonstrates the successful use of QSPR studies and Box–Behnken design in the preparation of nanotransfersomes for efficient delivery of FA in the management of psoriasis.

C-Myc Upregulated by High Glucose Inhibits HaCaT Differentiation by S100A6 Transcriptional Activation.

Keratinocyte differentiation dysfunction in diabetic skin is closely related to impaired skin barrier functions. We investigated the effects of c-Myc and S100A6 on Human immortal keratinocyte line (HaCaT) or keratinocyte differentiation and potential mechanisms. The expression levels of differentiation makers such as transglutaminase 1 (TGM1), loricrin (LOR), and keratin 1 (K1) were significantly reduced, while the expression of c-Myc was significantly increased in HaCaT cells cultured in high glucose and wound margin keratinocytes from diabetic rats and human patients. Overexpression of c-Myc caused differentiation dysfunction of HaCaT, while knocking down c-Myc promoted differentiation. High glucose increased the expression of c-Myc and inhibited differentiation in HaCaT cells by activating the WNT/β-catenin pathway. Moreover, inhibition of c-Myc transcriptional activity alleviated the differentiation dysfunction caused by high glucose or overexpression of c-Myc. c-Myc binds to the S100A6 promoter to directly regulate S100A6 expression and high glucose promoted S100A6 transcription. The expression of S100A6 was increased in HaCaT cultured with high glucose and wound margin keratinocytes from diabetic rats and human patients. However, the expression of S100A6 was decreased during normal HaCaT differentiation. HaCaT cells treated with S100A6 recombinant protein showed differentiation dysfunction. The expressions of TGM1, LOR and K1 in knockdown S100A6 HaCaT cells were higher than those in the control group. Overexpression of c-Myc or high glucose caused differentiation dysfunction of HaCaT cells, and was rescued by knocking down S100A6. These findings illustrate a new mechanism by which c-Myc upregulated by high glucose inhibits HaCaT differentiation by directly activating S100A6 transcription. Thus, c-Myc and S100A6 may be potential targets for the treatment of chronic diabetic wounds.

Pannexin 3 regulates skin development via Epiprofin

Pannexin 3 (Panx3), a member of the gap junction pannexin family is required for the development of hard tissues including bone, cartilage and teeth. However, the role of Panx3 in skin development remains unclear. Here, we demonstrate that Panx3 regulates skin development by modulating the transcription factor, Epiprofin (Epfn). Panx3−/− mice have impaired skin development and delayed hair follicle regeneration. Loss of Panx3 in knockout mice and suppression by shRNA both elicited a reduction of Epfn expression in the epidermis. In cell culture, Panx3 overexpression promoted HaCaT cell differentiation, cell cycle exit and enhanced Epfn expression. Epfn−/− mice and inhibition of Epfn by siRNA showed no obvious differences of Panx3 expression. Furthermore, Panx3 promotes Akt/NFAT signaling pathway in keratinocyte differentiation by both Panx3 ATP releasing channel and ER Ca2+ channel functions. Our results reveal that Panx3 has a key role factor for the skin development by regulating Epfn.

Biomolecular modifications during keratinocyte differentiation: Raman spectroscopy and chromatographic techniques.

From the basal layer until the stratum corneum, lipid and protein biomarkers associated with morphological changes denote keratinocyte differentiation and characterize each epidermis layer. Herein, we followed keratinocyte differentiation in the early stages using HaCaT cells over a period of two weeks by two complementary analytical techniques: Raman microspectroscopy and high-performance liquid chromatography coupled with high resolution mass spectrometry. A high concentration of calcium in the medium induced HaCaT cell differentiation in vitro. The results from both techniques underlined the keratinocyte passage from the granular layer (day 9) to the stratum corneum layer (day 13). After 13 days of differentiation, we observed a strong increase in the lipid content, decrease in proteins, decrease in DNA, and a decrease in glucosylceramides/ceramides and sphingomyelins/ceramides ratios.

Eucalyptus Citriodora Extract Regulates Cutaneous Homeostasis Including Immune Dysregulation and Skin Barrier Dysfunction Via the Modulation of Peroxisome Proliferator-Activated Receptor-β/δ (PPAR-β/δ) Pathway

Perturbation of cutaneous homeostasis including immune dysregulation and skin barrier dysfunction evokes skin disorders. In this study, we examined the effect of Eucalyptus citriodora (Euc-c) extract on cytokine production, cell proliferation and cell differentiation in HaCaT cells to elucidate its influence on cutaneous homeostasis. Euc-c suppressed significantly LPS-induced IL-6 and TNF-a-induced IL-8 production from HaCaT cells. Conversely IL-1ra production was significantly enhanced by Euc-c. The expressions of IVL, CERS3 and CERS4, keratinocyte differentiation markers, were upregulated to 3.1, 2.8 and 2.7-fold respectively by Euc-c treatment, compared to the control, while the proliferation was downregulated. The lipid contents in Euc-c-treated cells tended to increase, compared with non-treated cells. To explore the underlying mechanism of these effect, we next performed siRNA experiments against PPAR-b/d. Euc-c enhanced PPAR-b/d mRNA expression to 3.25-fold, while PPAR-b/d mRNA expression in transfected cells was suppressed. The expressions of IVL, CERS3 and CERS4 in transfected cells were suppressed to 1.48, 0.82 and 0.72-fold respectively, concomitant with suppression of PPAR-b/d mRNA expression. These results indicated that Euc-c exerts anti-inflammatory effects and regulates keratinocyte differentiation via the modulation of PPAR-b/d pathway. Therefore, the application of Euc-c is expected to exert beneficial effect on skin disorders evoked by perturbation of skin homeostasis.

Triptolide Inhibits the Proliferation of HaCaT Cells Induced by IL22 via Upregulating miR-181b-5p.

BackgroundEvidence has been shown that triptolide was effective in the treatment of psoriasis; however, the mechanisms remain poorly understood. Thus, this study aimed to investigate the role of triptolide on the proliferation and differentiation of HaCaT cells which are treated with IL22 to mimic abnormal proliferation/differentiation in keratinocyte of psoriasis.Materials and MethodsHaCaT cells were transfected with miR-181b-5p antagomir for 24 h, and then exposed to 10 μM Triptolide for 24 h, following by 100 ng/mL of IL22 for 24 h. In addition, the proliferation and cell cycle distribution in HaCaT cells were assessed by immunofluorescence or flow cytometry assays, respectively.ResultsTriptolide obviously upregulated the level of miR-181b-5p in HaCaT cells. In addition, triptolide significantly inhibited IL22-induced proliferation of HaCaT cells via inducing cell cycle arrest. Moreover, IL22 markedly inhibited the differentiation of HaCaT cells, and this phenomenon was reversed by triptolide treatment. In contrast, the effects of triptolide on the proliferation and differentiation in IL22-stimulated HaCaT cells were notably reversed by miR-181b-5p antagomir. Moreover, dual-luciferase assay showed that E2F5 was the direct target of miR-181b-5p in HaCaT cells. Meanwhile, upregulation of miR-181b-5p obviously decreased the level of E2F5 in HaCaT cells.ConclusionIn this study, we found that triptolide could inhibit the proliferation and promote the differentiation in IL22-stimulated keratinocytes via upregulating miR-181b-5p. These data indicated that triptolide may be a potential agent for the treatment of psoriasis.

The potential of Diosgenin in treating psoriasis: Studies from HaCaT keratinocytes and imiquimod-induced murine model

AimsPsoriasis is a cutaneous disease mainly characterized by keratinocyte hyperproliferation, abnormal epidermal differentiation, inflammation and angiogenesis. In this study, we aimed to report the therapeutic potential of Diosgenin on psoriasis-like models and explore the underlying mechanisms. Main methodsFor in vitro studies, we initially evaluated the bioeffects of Diosgenin on keratinocytes by detecting the cell viability, cell cycle and apoptosis in HaCaT cells. To mimic psoriatic conditions, we established hyperproliferative model by stimulating HaCaT cells with LPS/IL-22 and inflammatory model by LPS/TNF-α stimulation. Meanwhile, differentiation in HaCaT cells and angiogenesis in HUVECs/HMEC-1 were observed. The influence of Diosgenin on above-mentioned conditions was examined. For in vivo studies, we dosed imiquimod (IMQ) -induced mice with Diosgenin and conducted hematoxylin-eosin (HE), TUNEL assay and immunohistochemistry (IHC) to evaluate histological changes, apoptosis and the status of keratinocyte proliferation, epidermal differentiation, vascularity and cutaneous inflammatory cell infiltration respectively. Key findingsResults showed that Diosgenin inhibited HaCaT cell growth through cell cycle arrest and NFκB inhibition while induced apoptosis by regulating Caspase3, Bax and Bcl-2 protein expression. After Diosgenin treatment, NFκB nuclear translocation and IL-22 receptor dependent pathways were suppressed in LPS/IL-22 induced HaCaT cells respectively. Furthermore, Diosgenin downregulated proinflammatory cytokines through TLR4/Myd88 inhibition and upregulated several differentiation markers' expression in HaCaT cells. Additionally, Diosgenin inhibited vascular formation partially by reducing the VEGF-α expression in keratinocytes. In animal studies, Diosgenin attenuated psoriatic lesions on mice accordingly. SignificanceThis study suggests that Diosgenin might be a promising candidate for developing anti-psoriatic agents.

Fucoidan from Undaria pinnatifida Ameliorates Epidermal Barrier Disruption via Keratinocyte Differentiation and CaSR Level Regulation.

The epidermal barrier acts as a line of defense against external agents as well as helps to maintain body homeostasis. The calcium concentration gradient across the epidermal barrier is closely related to the proliferation and differentiation of keratinocytes (KCs), and the regulation of these two processes is the key to the repair of epidermal barrier disruption. In the present study, we found that fucoidan from Undaria pinnatifida (UPF) could promote the repair of epidermal barrier disruption in mice. The mechanistic study demonstrated that UPF could promote HaCaT cell differentiation under low calcium condition by up-regulating the expression of calcium-sensing receptor (CaSR), which could then lead to the activation of the Catenin/PLCγ1 pathway. Further, UPF could increase the expression of CaSR through activate the ERK and p38 pathway. These findings reveal the molecular mechanism of UPF in the repair of the epidermal barrier and provide a basis for the development of UPF into an agent for the repair of epidermal barrier repair.

Eucalyptus citriodora extract regulates cutaneous homeostasis including immune dysregulation and skin barrier dysfunction via the modulation of peroxisome proliferator-activated receptor-delta (PPAR-delata) pathway

Perturbation of cutaneous homeostasis including immune dysregulation and skin barrier dysfunction evokes skin disorders. In this study, we examined the effect of Eucalyptus citriodora (Euc-c) extract on cytokine production, cell proliferation and cell differentiation in HaCaT cells to elucidate its influence on cutaneous homeostasis. Euc-c suppressed significantly LPS-induced IL-6 and TNF-a-induced IL-8 production from HaCaT cells. Conversely IL-1ra production was significantly enhanced by Euc-c. The expressions of IVL, CERS3 and CERS4, keratinocyte differentiation markers, were upregulated to 3.1, 2.8 and 2.7-fold respectively by Euc-c treatment, compared to the control, while the proliferation was downregulated. The lipid contents in Euc-c-treated cells tended to increase, compared with non-treated cells. To explore the underlying mechanism of these effect, we next performed siRNA experiments against PPAR-b/d. Euc-c enhanced PPAR-b/d mRNA expression to 3.25-fold, while PPAR-b/d mRNA expression in transfected cells was suppressed. The expressions of IVL, CERS3 and CERS4 in transfected cells were suppressed to 1.48, 0.82 and 0.72-fold respectively, concomitant with suppression of PPAR-b/d mRNA expression. These results indicated that Euc-c exerts anti-inflammatory effects and regulates keratinocyte differentiation via the modulation of PPAR-b/d pathway. Therefore, the application of Euc-c is expected to exert beneficial effect on skin disorders evoked by perturbation of skin homeostasis.Key words: Eucalyptus citriodora, PPAR-b/d, inflammation, barrier function, cutaneous homeostasis

Jarid1b Promotes Epidermal Differentiation by Mediating Repression of Ship1 and Activation of AKT/Ovol1 Pathway

The role of epigenetic regulators in the control of epithelial cell differentiation is largely undefined. Our studies showed that H3K4me3 was decreased during epidermal differentiation. Unexpectedly, both expression of histone methyltransferases and demethylases over H3K4me3 were elevated upon differentiation, suggesting the crucial role of demethylases in controlling of differentiation. Next we found that the demethylase Jarid1b positively regulated epidermal differentiation. ShRNA-mediated depletion of Jarid1b delayed the calcium induced HaCaT cell differentiation in vitro and impaired differentiation layer formation in reconstituted epidermis in vivo. Conversely, overexpression of Jarid1b increased epidermal differentiation gene expression and inhibited cell proliferation and migration in culture. Mechanistically, we found that Jarid1b substantially increased mesenchymal-epithelial transition (MET) gene expression, among which Ovol1 positively regulated differentiation gene expression. In addition，Ovol1 expression was repressed by PI3K-AKT pathway inhibitors and suppressor Ship1 overexpression. Knockdown of Ship1 activated downstream PI3K-AKT pathway and enhanced Ovol1 expression in HaCaT cells. Importantly, we found that Jarid1b negatively regulated Ship1 expression, but not of Pten, via directly binding to its promoter to modulate H3K4me3 enrichment. Taken together, our results indentify an essential role for Jarid1b in regulation of Ship1/AKT/Ovol1 pathway to promote epithelial cell differentiation. Funding Statement: This work was supported by National Natural Science Foundation of China (81672662, 81701381, 81770978 and 81774248), Key Research and Development Program of Shandong Province (2017GSF18193, 2018GSF118012), Natural Science Foundation of Shandong Province (ZR2014CM040) and Shandong Provincial Education Department Program (J14LK02). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: All experimental protocols were approved by the ethics committee of animal care of Affiliated Hospital of Qingdao University. All animal studies were performed in strict accordance with the approved protocol.

Effect of metformin on proliferation and differentiation of HaCaT cells and secretion of inflammatory factors by HaCaT cells

Objective To evaluate the effect of metformin on the human keratinocyte line HaCaT, and to explore its molecular mechanism. Methods HaCaT cells were divided into several groups to be treated with metformin at different concentrations of 1, 2, 5, 10, 20, 50 mmol/L for 24, 48 and 72 hours. Cell counting kit-8 (CCK-8) assay was performed to evaluate the effect of metformin on the survival rate of HaCaT cells. After 48-hour treatment with metformin at concentrations of 0 (control group) , 0.5, 1, 2, 5, 10 mmol/L, flow cytometry was conducted to evaluate the effect of metformin on cell cycle and apoptosis. Western blot analysis was performed to determine the expression of cell proliferation- and differentiation-related proteins (keratin-16[K16], K17, K1, involucrin) , apoptosis-related proteins (Bax, Bcl-2) and AKT/mTOR/STAT3 pathway proteins. Enzyme-linked immunosorbent assay (ELISA) was conducted to detect the levels of interleukin (IL) -8, tumor necrosis factor (TNF) -α and IL-23 (inflammatory factors) in the culture supernatant of HaCaT cells. Statistical analysis was carried out with SPSS19.0 software using one-way analysis of variance for comparison of the above indices among the 0.5-, 1-, 2-, 5-, 10-mmol/L metformin groups and control group, repeated measures analysis of variance for comparisons among different time points or different metformin groups, least significant difference (LSD) -t test for multiple comparisons. Results CCK-8 assay showed that metformin had inhibitory effects on the proliferation of HaCaT cells (F= 116.87, P 0.05) . After 48-hour treatment with metformin at different concentrations, the levels of IL-8 and TNF-α in HaCaT cells significantly decreased (F= 33.89, 14.99 respectively, both P 0.05) . Along with the increase in the concentrations of metformin, the expression of p-AKT, p-mTOR and p-STAT3 significantly decreased (F= 11.38, 0.35, 4.38 respectively, all P 0.05) . Conclusion Metformin can inhibit the proliferation, promote the differentiation and apoptosis of HaCaT cells, and inhibit the secretion of inflammatory factors by regulating the AKT/mTOR/STAT3 signaling pathway. Key words: Metformin; Cell proliferation; Apoptosis; Cell differentiation; HaCaT cells

Regulation of 25-hydroxyvitamin D-1-hydroxylase and 24-hydroxylase in keratinocytes by PTH and FGF23.

Renal 25-hydroxyvitamin D-1α-hydroxylase (1αOHase, CYP27B1) and 24-hydroxylase (24OHase, CYP24A1) are tightly regulated. However, little is known about the regulation of 1α(OH)ase and 24(OH)ase in extrarenal tissue such as the epidermis. This study was to determine the roles of parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF 23) in the regulation of 1α(OH)ase and 24(OH)ase in epidermal keratinocytes as well as epidermal keratinocyte proliferation and differentiation. The results showed that PTH increased the protein level of 1α(OH)ase in human epidermal keratinocyte cell line HaCaT, but had no effect on the level of 24(OH)ase. The effect of PTH on 1α(OH)ase was blocked by the PKC inhibitor. Treatment with FGF23 decreased mRNA and protein levels of 1α(OH)ase and increased mRNA and protein levels of 24(OH)ase in HaCaT cells. The effect of FGF23 on 1α(OH)ase and 24(OH)ase was blocked by the mitogen-activated protein kinase/extracellular regulated protein kinase (MAPK/ERK) inhibitor. In addition, treatment with PTH enhanced levels of differentiation markers including keratin 1, involucrin, loricrin, and filaggrin but reduced levels of BrdU incorporation in HaCaT cells. These effects were inhibited by the PKC inhibitor. FGF23 enhanced proliferation of HaCaT cells, but reduced levels of early differentiation markers including keratin 1 and involucrin and enhanced levels of the later differentiation markers including loricrin and filaggrin. These results suggest that PTH stimulates 1α(OH)ase expression and differentiation of HaCaT cells and inhibits proliferation via PKC. The data also suggest that FGF23 inhibits 1α(OH)ase expression and stimulates 24(OH)ase expression via MAPK/ERK. In addition, FGF23 enhances proliferation and late differentiation and inhibits early differentiation of HaCaT keratinocytes.

637 Revesible differentiation in HaCaT cells

637 Revesible differentiation in HaCaT cells

Imiquimod-induced psoriasis-like inflammation in differentiated Human keratinocytes: Its evaluation using curcumin

Psoriasis is considered to be a systemic disease of immune dysfunction. It is still unclear what triggers the inflammatory cascade associated with psoriasis but recent evidences suggest the vital role of IL-23/IL-17A cytokine axis in etiology of psoriasis. Several studies have been conducted in psoriatic-like animal models but ethical issues and complexity surrounding it halts the screening of new anti-psoriatic drug candidates. Hence, in this study, we developed a new in-vitro model for psoriasis using imiquimod (IMQ) induced differentiated HaCaT cells which could be used for screening of new anti-psoriatic drug candidates. The differentiated HaCaT cells were treated with IMQ (100μM) to induce psoriatic like inflammation and its effect was investigated using a natural anti-psoriatic compound, curcumin. The proliferation of psoriatic-like cells was inhibited by curcumin at 25 and 50µM concentrations. The psoriatic-like cells decreased in number with increase in apoptotic and dead cells upon curcumin treatment. Curcumin inhibited the proliferation of IMQ-induced differentiated HaCaT cells (Psoriatic-like cells) by down-regulation of pro-inflammatory cytokines, interleukin-17, tumor necrosis factor-α, interferon-γ, and interleukin-6. Apart from this, curcumin significantly enhanced the skin-barrier function by up-regulation of involucrin (iNV) and filaggrin (FLG), the regulators of epidermal skin barrier. The IMQ-induced differentiated HaCaT in vitro model recapitulated some aspects of the psoriasis pathogenesis similar to murine model. Henceforth, we conclude that this model may be used for rapid screening of anti-psoriatic drug candidates and warrant further mechanistic studies.

Possible Involvement of F1F0-ATP synthase and Intracellular ATP in Keratinocyte Differentiation in normal skin and skin lesions

The F1F0-ATP synthase, an enzyme complex, is mainly located on the mitochondrial inner membrane or sometimes cytomembrane to generate or hydrolyze ATP, play a role in cell proliferation. This study focused on the role of F1F0-ATP synthase in keratinocyte differentiation, and its relationship with intracellular and extracellular ATP (InATP and ExATP). The F1F0-ATP synthase β subunit (ATP5B) expression in various skin tissues and confluence-dependent HaCaT differentiation models was detected. ATP5B expression increased with keratinocyte and HaCaT cell differentiation in normal skin, some epidermis hyper-proliferative diseases, squamous cell carcinoma, and the HaCaT cell differentiation model. The impact of InATP and ExATP content on HaCaT differentiation was reflected by the expression of the differentiation marker involucrin. Inhibition of F1F0-ATP synthase blocked HaCaT cell differentiation, which was associated with a decrease of InATP content, but not with changes of ExATP. Our results revealed that F1F0-ATP synthase expression is associated with the process of keratinocyte differentiation which may possibly be related to InATP synthesis.

Epidermal CFTR Suppresses MAPK/NF-κB to Promote Cutaneous Wound Healing

Background: CFTR is implicated in cutaneous wound healing although the underlying mechanisms are not fully understood. In other cell types, CFTR is reported to regulate MAPK/ NF-κB signaling. We undertook the present study to explore a possible role of CFTR in regulating MAPK/NF-κB during cutaneous wound healing. Methods& Results: The splint-excisional and incisional wound healing models were used in CFTR mutant (DF508) mice. The cell-scratch model was used in a human keratinocyte line, HaCaT, in conjunction with CFTR knockdown or overexpression. The epidermal inflammation, keratinocyte proliferation and differentiation, as well as MAPK/NF-κB signaling were examined. Inhibitors of MAPK/NF-κB were also used. Results: Both DF508 mice and HaCaT cells with CFTR knockdown exhibited delayed cutaneous wound healing with exuberant inflammation, increased proliferation and aberrant differentiation. Knockdown of CFTR in HaCaT cells resulted in phosphorylation of ERK, p38 and IκBα. The disturbance of inflammation, proliferation and differentiation in HaCaT cells were reversed by CFTR overexpression or inhibition of MAPK or NF-κB. Conclusion: CFTR plays a role in suppressing MAPK/NF-κB to relieve inflammation, reduce proliferation and promote differentiation of keratinocytes, and thus promotes cutaneous wound healing.