Involucrin Gene Expression Research Articles

개머루덩굴 추출물의 자극에 의한 피부장벽 개선 효과

The skin is the largest organ that blocks invasion to the outside. Due to external stimuli, the skin barrier collapses and immune function abnormalities occur. This leads to skin diseases such as atopic dermatitis and psoriasis. Through this study, we attempted to develop a natural material that can be used for various dermatitis diseases. In this study, the gene expression of skin barrier-related proteins was confirmed using tumor necrosis factor-α/interferon-γ-stimulated HaCaT cells through real-time PCR. As a result, it was confirmed that the gene expression of skin barrier-related proteins and ceramide-related proteins was improved by the ABE-M. In this study, it was confirmed that the gene expression of filaggrin, involucrin, and loricrin, which are skin barrier-related proteins, was improved by ABE-M, and it was confirmed that the gene expression of the tight junction-related proteins, occludin and zonula occludens proteins-1, was also increased. As such, it is expected that ABE-M can be used as a material for skin inflammation such as atopic dermatitis.

Laser Treatment of Hypertrophic Scar in a Porcine Model Induces Change to Epidermal Histoarchitecture That Correlates to Improved Epidermal Barrier Function.

Mechanisms and timing of hypertrophic scar (HTS) improvement with laser therapy are incompletely understood. Epidermal keratinocytes influence HTS through paracrine signaling, yet they are understudied compared to fibroblasts. It was hypothesized that fractional ablative CO2 laser scar revision (FLSR) would change the fibrotic histoarchitecture of the epidermis in HTS. Duroc pigs (n = 4 FLSR and n = 4 controls) were injured and allowed to form HTS. HTS and normal skin (NS) were assessed weekly by noninvasive skin probes measuring trans-epidermal water loss (TEWL) and biopsy collection. There were 4 weekly FLSR treatments. Immediate laser treatment began on day 49 postinjury (just after re-epithelialization), and early treatment began on day 77 postinjury. Punch biopsies from NS and HTS were processed and stained with H&E. Epidermal thickness and rete ridge ratios (RRR) were measured. Gene and protein expression of involucrin (IVL) and filaggrin (FIL) were examined through qRT-PCR and immunofluorescent (IF) staining. After treatment, peeling sheets of stratum corneum were apparent which were not present in the controls. TEWL was increased in HTS vs NS at day 49, indicating decreased barrier function (P = .05). In the immediate group, TEWL was significantly decreased at week 4 (P < .05). The early group was not significantly different from NS at the prelaser timepoint. After four sessions, the epidermal thickness was significantly increased in treated scars in both FLSR groups (immediate: P < .01 and early: P < .001, n = 8 scars). Early intervention significantly increased RRR (P < .05), and immediate treatment trended toward an increase. There was no increase in either epidermal thickness or RRR in the controls. In the immediate intervention group, there was increased IVL gene expression in HTS vs NS that decreased after FLSR. Eight scars had upregulated gene expression of IVL vs NS levels pretreatment (fold change [FC] > 1.5) compared to four scars at week 4. This was confirmed by IF where IVL staining decreased after FLSR. FIL gene expression trended towards a decrease in both interventions after treatment. Changes in epidermal HTS histoarchitecture and expression levels of epidermal differentiation markers were induced by FLSR. The timing of laser intervention contributed to differences in TEWL, epidermal thickness, and RRR. These data shed light on the putative mechanisms of improvement seen after FLSR treatment. Resolution of timing must be further explored to enhance efficacy. An increased understanding of the difference between the natural history of HTS improvement over time and interventional-induced changes will be critical to justifying the continued approved usage of this treatment.

Ozonized glycerin (OG)-based cosmetic products lighten age spots on human facial skin.

BackgroundFew cosmetic ingredients are shown to be able to safely remove or lighten facial dark spots once they have formed. OG has been reported to possess oxidation power and exhibit various biological activities such as antibacterial, antiviral, and wound healing promotion.AimsThis study aimed to clarify the effects of OG on human skin, especially on age spots on the face.MethodsOG formulations (80 and 800 ppm) were mixed with synthetic melanin in vitro for 4 weeks and then assessed for its ability to degrade the melanin. OG also investigated its effect on gene expression of keratinocyte differentiation markers in vitro to explore the cell maturation. In clinical study for the evaluation of effects of OG formulations on age spots on facial skin, 48 women were measured for the melanin content of them by a Mexameter at 4 and 8 weeks after daily twice application of OG formulations. Adverse events were monitored during the study.ResultsBoth OG formulations showed direct melanin degradation in a time‐dependent manner, with significant effects observed as early as 6 h. OG formulation at 800 ppm showed higher activity than OG formulation at 80 ppm, and the amount of melanin was decreased by about 40% on Day 14 of the mixing reaction. Differentiation marker studies using human keratinocytes showed that the gene expression of involucrin and serine palmitoyltransferase was upregulated by OG, which was almost equivalent concentration to OG formulation 80 ppm, suggesting that OGs can enhance turnover of the skin epidermis. In clinical study, OG formulations 80 and 800 ppm showed larger decreases in melanin contents at 8 weeks compared with those at 4 weeks and their mean values of △melanin index were −16.7 and −15.2, respectively. Statistically significant differences were detected against respective controls. Number of subjects with a decrease in melanin index from baseline to 4 or 8 weeks increased in both OG formulations 80 and 800 ppm, especially prominent at 8 weeks. There were no adverse events related to treatments of OG 80 and 800 ppm during the study.ConclusionThe result indicated that applications of OG formulations are safe and effective in lightening age spots on the facial skin.

614 Laser-treatment of Hypertrophic Scar Induces Change to Epidermal Histoarchitecture Correlating to Improved Epidermal Barrier Function

IntroductionMechanisms and timing of hypertrophic scar (HTS) improvement with laser therapy are incompletely understood. Epidermal keratinocytes influence HTS through paracrine signaling, yet they are understudied compared to fibroblasts. It was hypothesized that fractional ablative CO2 laser scar revision (FLSR) would change the fibrotic histoarchitecture of the epidermis in HTS.MethodsDuroc pigs (n=4 FLSR and n=4 controls) were injured and allowed to form HTS. HTS and normal skin (NS) were assessed weekly by non-invasive skin probes measuring trans-epidermal water loss (TEWL) and biopsy collection. There were 4 weekly FLSR treatments. Early laser treatment began on day 49, and late began on day 77. Punch biopsies from NS and HTS were processed and stained with H&E. Image J was used to obtain epidermal thickness and rete ridge ratios (RRR). Gene and protein expression of involucrin (IVL) was examined through qRT-PCR and immunofluorescent (IF) staining.ResultsAfter treatment, peeling sheets of stratum corneum were apparent which were not present in the controls. TEWL was increased in HTS vs. NS at day 49 indicating decreased barrier function (42.2±8.0 vs. 22.0±4.62g/m2h, p=0.05). In the early group, TEWL was significantly decreased at week 4 to 16.4±3.5 g/m2h (p< 0.05). The late group was not significantly altered from NS at the pre-laser timepoint (day 77=12.1±1.99 g/m2h). Hence, there was no decrease in TEWL post-FLSR. After 4 sessions, epidermal thickness was significantly increased in treated scars in both FLSR groups (early:pre=85.6±6.8 vs. week 4=115.2±12.0 µm, p< 0.01) and (late:pre=75.2±6.6 vs. week 4=125.7±12.0 µm p< 0.001, n=8 scars,). There was no increase in controls. Late intervention significantly increased RRR (pre=1.3±0.1 vs. week 4=1.9±0.1, n=8 scars, p< 0.05), and early treatment trended towards increase (pre=1.17±0.05 vs. week 4=1.4 + 0.1). There was no increase in controls. There was increased IVL gene expression in HTS vs. NS that decreased after FLSR. Eight scars had up-regulated gene expression of IVL vs. NS levels pre-treatment (FC >1.5) compared to 4 scars at week 4. This was confirmed by IF where IVL staining decreased at week 4.ConclusionsChanges in epidermal HTS histoarchitecture and expression levels of epidermal differentiation markers were induced by FLSR. The timing of laser intervention contributed to differences in TEWL, epidermal thickness, and RRR.

Lithospermum erythrorhizon Alleviates Atopic Dermatitis-like Skin Lesions by Restoring Immune Balance and Skin Barrier Function in 2.4-Dinitrochlorobenzene-Induced NC/Nga Mice.

The incidence of atopic dermatitis (AD), a disease characterized by an abnormal immune balance and skin barrier function, has increased rapidly in developed countries. This study investigated the anti-atopic effect of Lithospermum erythrorhizon (LE) using NC/Nga mice induced by 2,4-dinitrochlorobenzene. LE reduced AD clinical symptoms, including inflammatory cell infiltration, epidermal thickness, ear thickness, and scratching behavior, in the mice. Additionally, LE reduced serum IgE and histamine levels, and restored the T helper (Th) 1/Th2 immune balance through regulation of the IgG1/IgG2a ratio. LE also reduced the levels of AD-related cytokines and chemokines, including interleukin (IL)-1β, IL-4, IL-6, tumor necrosis factor-α (TNF-α), thymic stromal lymphopoietin, thymus and activation-regulated chemokine, macrophage-derived chemokine, regulated on activation, normal T cell expressed and secreted, and monocyte chemoattractant protein-1 in the serum. Moreover, LE modulated AD-related cytokines and chemokines expressed and secreted by Th1, Th2, Th17, and Th22 cells in the dorsal skin and splenocytes. Furthermore, LE restored skin barrier function by increasing pro-filaggrin gene expression and levels of skin barrier-related proteins filaggrin, involucrin, loricrin, occludin, and zonula occludens-1. These results suggest that LE is a potential therapeutic agent that can alleviate AD by modulating Th1/Th2 immune balance and restoring skin barrier function.

313 In vivo enhancer deletion in the EDC results in decreased proximal involucrin and Lce6a target gene expression marked by less accessible chromatin

313 In vivo enhancer deletion in the EDC results in decreased proximal involucrin and Lce6a target gene expression marked by less accessible chromatin

γ-Propoxy-Sulfo-Lichenan Induces In Vitro Cell Differentiation of Human Keratinocytes.

Background: As non-cellulosic β-d-glucans are known to exert wound-healing activity by triggering keratinocytes into cellular differentiation, the functionality of a semisynthetic lichenan-based polysaccharide on skin cell physiology was investigated. Methods: γ-Propoxy-sulfo-lichenan (γ-PSL, molecular weight 52 kDa, β-1,3/1,4-p-d-Glucose, degree of substitution 0.7) was prepared from lichenan. Differentiation of primary human keratinocytes was assayed by the protein analysis of differentiation specific markers and by gene expression analysis (qPCR). The gene array gave insight into the cell signaling induced by the polysaccharide. Results: γ-PSL (1 to 100 μg/mL) triggered keratinocytes, in a concentration-dependent manner, into the terminal differentiation, as shown by the increased protein expression of cytokeratin 1 (KRT1). Time-dependent gene expression analysis proved differentiation-inducing effects, indicating strong and fast KRT1 gene expression, while KRT10 expression showed a maximum after 12 to 24 h, followed by downregulation to the basal level. Involucrin gene expression was only changed to a minor extent, which was similar to loricrin and transglutaminase. Gene array indicated the influence of γ-PSL on MAP kinase and TGF-β mediated signaling towards keratinocyte differentiation. Conclusion: The propoxylated lichenan may improve wound healing by topical application to promote the terminal barrier formation of keratinocytes.

β-1,3/1,4-Glucan Lichenan from Cetraria islandica (L.) ACH. induces cellular differentiation of human keratinocytes

Lichenan (molecular weight 275 kDa, β-D-1,3/1,4-glucopyranose ratio 1:3) from Cetraria islandica at a concentration of 100 μg/mL induced terminal cellular differentiation of primary human keratinocytes as demonstrated by immunofluorescence staining using cytokeratin 10 and involucrin as marker proteins. Lichenan-derived oligosaccharides (DP3 to 8), obtained by acid-catalyzed partial hydrolysis of the polymer, did not influence cellular differentiation. Cytokeratin, filaggrin, involucrin, loricrin and transglutaminase gene expression as typical differentiation markers was increased by lichenan in a time-dependent manner. Lichenan upregulated gene cluster which were mostly related to cellular differentiation with focus on MAPK signaling as was shown by Whole Human Genome Microarray. These gene expression data from the array experiments were subsequently confirmed by qPCR for selected genes. For identification of the molecular binding structures of lichenan 1- and 2-D PAGE of keratinocyte protein membrane preparations was performed, followed by blotting with FITC-labeled lichenan and subsequent mass spectrometric identification of the pinpointed proteins. Epidermal growth factor receptor (EGFR) and integrin β4, both proteins being strongly involved in induction of keratinocyte differentiation were identified. In addition, protein disulfide isomerase A3 (PDIA3) showed strong binding to FITC-lichenan, indicating this enzyme to be an intracellular target of the glucan for induction of the cellular differentiation of keratinocytes. As lichenan did not influence the EGFR phosphorylation and the phosphorylation of CREB transcription factor but strongly interacted with cytosolic proteins it is hypothized that the glucan may interact with EGFR and is subsequently internalized into the cell via endosomal uptake, interacting with PDIA3, which again alters TGFβ1 signaling towards keratinocyte differentiation.

829 The EDC enhancer 923 is required for Ivl, Smcp, and Lce6a gene expression and chromatin accessibility

The mechanisms by which gene expression is activated at the chromatin level are poorly understood. The Epidermal Differentiation Complex (EDC), locus whose genes are expressed upon keratinocyte differentiation, provides a unique opportunity to address this question. Previously we identified a role for the epidermal-specific 923 enhancer in dynamic chromatin remodeling. Here we sought to investigate whether 923 is necessary for EDC activation using CRISPR/Cas9-mediated deletion. 923-/- KO mice (both 923delA and 923delB) appeared phenotypically normal under homeostatic conditions. However, transcriptional profiling of 923delA-/- embryonic skin captured during epidermal development (E14.5–NB) revealed decreased proximal gene expression of involucrin (Ivl), late cornified envelope 6a (Lce6a), and sperm mitochondrial-associated cysteine rich protein (Smcp). The requirement for the 923 enhancer was further validated in 923delB-/- newborn keratinocytes. ATAC-seq in 923delA-/- and wild-type littermate newborn keratinocytes further identified more closed chromatin (fewer ATAC-seq peaks) observed at the 5’ and 3’ ends of the EDC in 923delA-/-, indicating a requirement for the 923 enhancer for chromatin organization at the EDC boundaries. Male fertility defects associated with decreased 923 enhancer target gene Smcp expression in 923delA-/- male testes were observed with reduced progeny and slower sperm motility. Our genomic study reporting in vivo functional effect for the deletion of an epidermal-specific enhancer, identifies a requirement for 923 in proximal Ivl, Lce6a, and Smcp target gene expression, higher level chromatin accessibility in the EDC, and downstream male fertility function for its Smcp target gene, thus addressing a knowledge gap for enhancer function in chromatin level gene expression.

724 Recent and independent emergences of population-specific enhancer eQTLs that modulate Involucrin gene expression for human skin barrier calibration

724 Recent and independent emergences of population-specific enhancer eQTLs that modulate Involucrin gene expression for human skin barrier calibration

Platelet-Released Growth Factors Induce Differentiation of Primary Keratinocytes.

Autologous thrombocyte concentrate lysates, for example, platelet-released growth factors, (PRGFs) or their clinically related formulations (e.g., Vivostat PRF®) came recently into the physicians' focus as they revealed promising effects in regenerative and reparative medicine such as the support of healing of chronic wounds. To elucidate the underlying mechanisms, we analyzed the influence of PRGF and Vivostat PRF on human keratinocyte differentiation in vitro and on epidermal differentiation status of skin wounds in vivo. Therefore, we investigated the expression of early (keratin 1 and keratin 10) and late (transglutaminase-1 and involucrin) differentiation markers. PRGF treatment of primary human keratinocytes decreased keratin 1 and keratin 10 gene expression but induced involucrin and transglutaminase-1 gene expression in an epidermal growth factor receptor- (EGFR-) dependent manner. In concordance with these results, microscopic analyses revealed that PRGF-treated human keratinocytes displayed morphological features typical of keratinocytes undergoing terminal differentiation. In vivo treatment of artificial human wounds with Vivostat PRF revealed a significant induction of involucrin and transglutaminase-1 gene expression. Together, our results indicate that PRGF and Vivostat PRF induce terminal differentiation of primary human keratinocytes. This potential mechanism may contribute to the observed beneficial effects in the treatment of hard-to-heal wounds with autologous thrombocyte concentrate lysates in vivo.

Electrospun tilapia collagen nanofibers accelerating wound healing via inducing keratinocytes proliferation and differentiation

The development of biomaterials with the ability to induce skin wound healing is a great challenge in biomedicine. In this study, tilapia skin collagen sponge and electrospun nanofibers were developed for wound dressing. The collagen sponge was composed of at least two α-peptides. It did not change the number of spleen-derived lymphocytes in BALB/c mice, the ratio of CD4+/CD8+ lymphocytes, and the level of IgG or IgM in Sprague-Dawley rats. The tensile strength and contact angle of collagen nanofibers were 6.72±0.44MPa and 26.71±4.88°, respectively. They also had good thermal stability and swelling property. Furthermore, the nanofibers could significantly promote the proliferation of human keratinocytes (HaCaTs) and stimulate epidermal differentiation through the up-regulated gene expression of involucrin, filaggrin, and type I transglutaminase in HaCaTs. The collagen nanofibers could also facilitate rat skin regeneration. In the present study, electrospun biomimetic tilapia skin collagen nanofibers were succesfully prepared, were proved to have good bioactivity and could accelerate rat wound healing rapidly and effectively. These biological effects might be attributed to the biomimic extracellular matrix structure and the multiple amino acids of the collagen nanofibers. Therefore, the cost-efficient tilapia collagen nanofibers could be used as novel wound dressing, meanwhile effectively avoiding the risk of transmitting animal disease in the future clinical apllication.

Luteolin induces caspase-14-mediated terminal differentiation in human epidermal keratinocytes.

Recent studies have demonstrated the role of caspase-14 in terminally differentiated keratinocytes, and its expression may decrease the magnitude of tumors in the epidermis. In the present study, we assessed the potential of luteolin (LUT) to elicit the expression of caspase-14 in terminal differentiation of human keratinocytes. The semi-qualitative RT-PCR data revealed a significant level of caspase-14 expression in LUT-treated human immortalized keratinocytes (HaCaT) with respect to untreated cells. The quantitative data (ELISA) further supported the potency of LUT to induce caspase-14 expression at 3.19ng/ml when compared to 1.29ng/ml of vitamin D3 (positive control). Further, the enhanced expression of human involucrin gene in LUT-treated HaCaT cells confirmed its ability to drive terminal differentiation in these cells. These preliminary results provide first-hand information about the in vitro potential of LUT to elicit the expression of caspase-14, thereby inducing terminal differentiation in human keratinocytes.

Retraction of "Development of Biomimetic Tilapia Collagen Nanofibers for Skin Regeneration through Inducing Keratinocytes Differentiation and Collagen Synthesis of Dermal Fibroblasts".

In this study, tilapia skin collagen sponge and electrospun nanofibers were developed for wound dressing. The collagen sponge was composed of at least two α-peptides, and its denaturation temperature was 44.99 °C. It did not change the number of spleen-derived lymphocytes in BALB/c mice, the ratio of CD4+/CD8+ lymphocytes, and the level of IgG or IgM in Sprague–Dawley rat. The contact angle, tensile strength, and weight loss temperature of collagen nanofibers were 21.2°, 6.72 ± 0.44 MPa, and 300 °C, respectively. The nanofibers could promote the viabilities of human keratinocytes (HaCaTs) and human dermal fibroblasts (HDFs), inducing epidermal differentiation through the gene expression of involucrin, filaggrin, and type I transglutaminase of HaCaTs, and they could also accelerate migration of HaCaTs with the expression of matrix metalloproteinase-9 and transforming growth factor-β1 (TGF-β1). Besides, the nanofibers could upregulate the protien level of Col-I in HDFs both via a direct effect and TGF-β1 se...

Thymic stromal lymphopoietin downregulates filaggrin expression by signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK) phosphorylation in keratinocytes

Thymic stromal lymphopoietin downregulates filaggrin expression by signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK) phosphorylation in keratinocytes

Development of biomimetic tilapia collagen nanofibers for skin regeneration through inducing keratinocytes differentiation and collagen synthesis of dermal fibroblasts.

In this study, tilapia skin collagen sponge and electrospun nanofibers were developed for wound dressing. The collagen sponge was composed of at least two α-peptides, and its denaturation temperature was 44.99 °C. It did not change the number of spleen-derived lymphocytes in BALB/c mice, the ratio of CD4+/CD8+ lymphocytes, and the level of IgG or IgM in Sprague-Dawley rat. The contact angle, tensile strength, and weight loss temperature of collagen nanofibers were 21.2°, 6.72±0.44 MPa, and 300 °C, respectively. The nanofibers could promote the viabilities of human keratinocytes (HaCaTs) and human dermal fibroblasts (HDFs), inducing epidermal differentiation through the gene expression of involucrin, filaggrin, and type I transglutaminase of HaCaTs, and they could also accelerate migration of HaCaTs with the expression of matrix metalloproteinase-9 and transforming growth factor-β1 (TGF-β1). Besides, the nanofibers could upregulate the protien level of Col-I in HDFs both via a direct effect and TGF-β1 secreted from HaCaTs, thus facilitating the formation of collagen fibers. Furthermore, the collagen nanofibers stimulated the skin regeneration rapidly and effectively in vivo. These biological effects could be explained as the contributions from the biomimic extracellular cell matrix structure, hydrophilicity, and the multiple amino acids of the collagen nanofibers.

P38δ Regulates p53 to Control p21Cip1 Expression in Human Epidermal Keratinocytes

PKCδ suppresses keratinocyte proliferation via a mechanism that involves increased expression of p21(Cip1). However, the signaling mechanism that mediates this regulation is not well understood. Our present studies suggest that PKCδ activates p38δ leading to increased p21(Cip1) promoter activity and p21(Cip1) mRNA/protein expression. We further show that exogenously expressed p38δ increases p21(Cip1) mRNA and protein and that p38δ knockdown or expression of dominant-negative p38 attenuates this increase. Moreover, p53 is an intermediary in this regulation, as p38δ expression increases p53 mRNA, protein, and promoter activity, and p53 knockdown attenuates the activation. We demonstrate a direct interaction of p38δ with PKCδ and MEK3 and show that exogenous agents that suppress keratinocyte proliferation activate this pathway. We confirm the importance of this regulation using a stratified epidermal equivalent model, which mimics in vivo-like keratinocyte differentiation. In this model, PKCδ or p38δ knockdown results in reduced p53 and p21(Cip1) levels and enhanced cell proliferation. We propose that PKCδ activates a MEKK1/MEK3/p38δ MAPK cascade to increase p53 levels and p53 drives p21(Cip1) gene expression.

Alterations of keratins, involucrin and filaggrin gene expression in canine atopic dermatitis

Canine atopic dermatitis (CAD) is a common allergic skin disease in dogs, associated with a defective epidermal barrier. In this study we investigated the alterations in skin keratinocyte proliferation and differentiation in CAD by quantitative reverse transcription-polymerase chain reaction. Gene expression of keratin (KRT) markers of proliferative and differentiated keratinocytes, together with that of cornified envelope proteins, involucrin (IVL) and filaggrin (FLG), were evaluated. An upregulation of KRT5 and KRT17 in both lesional and non-lesional AD skin was observed (p<0.05) whereas KRT2e, KRT14, IVL and FLG expression were significantly increased only in lesional AD skin (p<0.05). Additionally, the expression levels of KRT5, KRT14, KRT17 and IVL in CAD were strongly correlated. In conclusion, the expression of the majority of the studied keratins, as well as IVL and FLG is increased in CAD with close correlation between the proliferative keratins. This is the first report of a correlation of KRT and IVL genes with CAD.

Protein Arginine Methyltransferase 5 (PRMT5) Signaling Suppresses Protein Kinase Cδ- and p38δ-dependent Signaling and Keratinocyte Differentiation

PKCδ is a key regulator of keratinocyte differentiation that activates p38δ phosphorylation leading to increased differentiation as measured by an increased expression of the structural protein involucrin. Our previous studies suggest that p38δ exists in association with protein partners. A major goal is to identify these partners and understand their role in regulating keratinocyte differentiation. In this study we use affinity purification and mass spectrometry to identify protein arginine methyltransferase 5 (PRMT5) as part of the p38δ signaling complex. PRMT5 is an arginine methyltransferase that symmetrically dimethylates arginine residues on target proteins to alter target protein function. We show that PRMT5 knockdown is associated with increased p38δ phosphorylation, suggesting that PRMT5 impacts the p38δ signaling complex. At a functional level we show that PRMT5 inhibits the PKCδ- or 12-O-tetradecanoylphorbol-13-acetate-dependent increase in human involucrin expression, and PRMT5 dimethylates proteins in the p38δ complex. Moreover, PKCδ expression reduces the PRMT5 level, suggesting that PKCδ activates differentiation in part by reducing PRMT5 level. These studies indicate antagonism between the PKCδ and PRMT5 signaling in control of keratinocyte differentiation.

Chemical synthesis of 20 S-hydroxyvitamin D3, which shows antiproliferative activity

20 S-hydroxyvitamin D3 (20 S-(OH)D3), an in vitro product of vitamin D3 metabolism by the cytochrome P450scc, was recently isolated, identified and shown to possess antiproliferative activity without inducing hypercalcemia. The enzymatic production of 20 S-(OH)D3 is tedious, expensive, and cannot meet the requirements for extensive chemical and biological studies. Here we report for the first time the chemical synthesis of 20 S-(OH)D3 which exhibited biological properties characteristic of the P450scc-generated compound. Specifically, it was hydroxylated to 20,23-dihydroxyvitamin D3 and 17,20-dihydroxyvitamin D3 by P450scc and was converted to 1α,20-dihydroxyvitamin D3 by CYP27B1. It inhibited proliferation of human epidermal keratinocytes with lower potency than 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in normal epidermal human keratinocytes, but with equal potency in immortalized HaCaT keratinocytes. It also stimulated VDR gene expression with similar potency to 1,25(OH)2D3, and stimulated involucrin (a marker of differentiation) and CYP24 gene expression, showing a lower potency for the latter gene than 1,25(OH)2D3. Testing performed with hamster melanoma cells demonstrated a dose-dependent inhibition of cell proliferation and colony forming capabilities similar or more pronounced than those of 1,25(OH)2D3. Thus, we have developed a chemical method for the synthesis of 20 S-(OH)D3, which will allow the preparation of a series of 20 S-(OH)D3 analogs to study structure–activity relationships to further optimize this class of compound for therapeutic use.