Fibroblast Growth Factor Receptor Inhibitors Decrease Proliferation of Melanoma Cell Lines and Their Activity Is Modulated by Vitamin D.

Regardless of the unprecedented progress in malignant melanoma treatment strategies and clinical outcomes of patients during the last twelve years, this skin cancer remains the most lethal one. We have previously documented that vitamin D and its low-calcaemic analogues enhance the anticancer activity of drugs including a classic chemotherapeutic-dacarbazine-and an antiangiogenic VEGFRs inhibitor-cediranib. In this study, we explored the response of A375 and RPMI7951 melanoma lines to CPL304110 (CPL110), a novel selective inhibitor of fibroblast growth factor receptors (FGFRs), and compared its efficacy with that of AZD4547, the first-generation FGFRs selective inhibitor. We also tested whether 1,25(OH)2D3, the active form of vitamin D, modulates the response of the cells to these drugs. CPL304110 efficiently decreased the viability of melanoma cells in both A375 and RPMI7951 cell lines, with the IC50 value below 1 µM. However, the metastatic RPMI7951 melanoma cells were less sensitive to the tested drug than A375 cells, isolated from primary tumour site. Both tested FGFR inhibitors triggered G0/G1 cell cycle arrest in A375 melanoma cells and increased apoptotic/necrotic SubG1 fraction in RPMI7951 melanoma cells. 1,25(OH)2D3 modulated the efficacy of CPL304110, by decreasing the IC50 value by more than 4-fold in A375 cell line, but not in RPMI7951 cells. Further analysis revealed that both inhibitors impact vitamin D signalling to some extent, and this effect is cell line-specific. On the other hand, 1,25(OH)2D3, have an impact on the expression of FGFR receptors and phosphorylation (FGFR-Tyr653/654). Interestingly, 1,25(OH)2D3 and CPL304110 co-treatment resulted in activation of the ERK1/2 pathway in A375 cells. Our results strongly suggested possible crosstalk between vitamin D-activated pathways and activity of FGFR inhibitors, which should be considered in further clinical studies.

Different impact of vitamin D on mitochondrial activity and morphology in normal and malignant keratinocytes, the role of genomic pathway.

Deregulation of mitochondria activity is one of the hallmarks of cancerogenesis and an important target for cancer therapy. Therefore, we compared the impact of an active form of vitamin D3 (1,25(OH)2D3) on mitochondrial morphology and bioenergetics in human squamous cell carcinoma (A431) and immortalized HaCaT keratinocytes. It was shown that mitochondria of cancerous A431 cells differ from that observed in HaCaT keratinocytes in terms of network, morphology, bioenergetics, glycolysis, and mitochondrial DNA copy number, while treatment of A431 with 1,25(OH)2D3 partially eliminates these differences. Furthermore, mitochondrial membrane potential, basal respiration, and mitochondrial reactive oxygen species production were decreased in A431 cells treated with 1,25(OH)2D3. Additionally, the expression and protein level of mitophagy marker PINK1 was significantly increased in A431 1,25(OH)2D3 treated cells, but not observed in treated HaCaT cells. Knockout of VDR (vitamin D receptor) or RXRA (binding partner retinoid X receptor) partially altered mitochondrial morphology and function as well as mitochondrial response to 1,25(OH)2D3. Transcriptomic analysis on A431 cells treated with 1,25(OH)2D3 revealed modulation of expression of several mitochondrial-related genes involved in mitochondrial depolarization, mitochondrial protein translation (i.e. LYRM9, MARS2), and fusion-fission (OPA1, FIS1, MFN1 and 2), however, none of the genes coded by mitochondrial DNA was affected. Interestingly, in silico analyses of nuclear-encoded mitochondrial genes revealed that they are rather activated by the secondary genomic response to 1,25(OH)2D3. Taken together, 1,25(OH)2D3 remodels mitochondrial architecture and bioenergetics through VDR-dependent and only partially RXRA-dependent activation of the genomic pathway, thus outlining a new perspective for anticancer properties of vitamin D3 in relation to mitochondria in squamous cell carcinoma.

Dissection of an impact of VDR and RXRA on the genomic activity of 1,25(OH)2D3 in A431 squamous cell carcinoma.

BACKGROUND: Human skin is the natural source, place of metabolism, and target for vitamin D3. The classical active form of vitamin D3, 1,25(OH)2D3, expresses pluripotent properties and is intensively studied in cancer prevention and therapy. To define the specific role of vitamin D3 receptor (VDR) and its co-receptor retinoid X receptor alpha (RXRA) in genomic regulation, VDR or RXRA genes were silenced in the squamous cell carcinoma cell line A431 and treated with 1,25(OH)2D3 at long incubation time points 24 h/72 h. Extending the incubation time of A431 WT (wild-type) cells with 1,25(OH)2D3 resulted in a two-fold increase in DEGs (differentially expressed genes) and a change in the amount of downregulated from 37% to 53%. VDR knockout led to a complete loss of 1,25(OH)2D3-induced genome-wide gene regulation at 24 h time point, but after 72 h, 20 DEGs were found, of which 75% were downregulated, and most of them belonged to the gene ontology group "immune response". This may indicate the existence of an alternative, secondary response to 1,25(OH)2D3. In contrast, treatment of A431 ΔRXRA cells with 1,25(OH)2D3 for 24 h only partially affected DEGs, suggesting RXRA-independent regulation. Interestingly, overexpression of classic 1,25(OH)2D3 targets, like CYP24A1 (family 24 of subfamily A of cytochrome P450 member 1) or CAMP (cathelicidin antimicrobial peptide) was found to be RXRA-independent. Also, immunofluorescence staining of A431 WT cells revealed partial VDR/RXRA colocalization after 24 h and 72 h 1,25(OH)2D3 treatment. Comparison of transcriptome changes induced by 1,25(OH)2D3 in normal keratinocytes vs. cancer cells showed high cell type specific expression pattern with only a few genes commonly regulated by 1,25(OH)2D3. Activation of the genomic pathway at least partially reversed the expression of cancer-related genes, forming a basis for anti-cancer activates of 1,25(OH)2D3. In summary, VDR or RXRA independent genomic activities of 1,25(OH)2D3 suggest the involvement of alternative factors, opening new challenges in this field.

Protein Disulfide Isomerase Family A Member 3 Knockout Abrogate Effects of Vitamin D on Cellular Respiration and Glycolysis in Squamous Cell Carcinoma.

PDIA3 is an endoplasmic reticulum disulfide isomerase, which is involved in the folding and trafficking of newly synthesized proteins. PDIA3 was also described as an alternative receptor for the active form of vitamin D (1,25(OH)2D3). Here, we investigated an impact of PDIA3 in mitochondrial morphology and bioenergetics in squamous cell carcinoma line A431 treated with 1,25(OH)2D3. It was observed that PDIA3 deletion resulted in changes in the morphology of mitochondria including a decrease in the percentage of mitochondrial section area, maximal diameter, and perimeter. The 1,25(OH)2D3 treatment of A431∆PDIA3 cells partially reversed the effect of PDIA3 deletion increasing aforementioned parameters; meanwhile, in A431WT cells, only an increase in mitochondrial section area was observed. Moreover, PDIA3 knockout affected mitochondrial bioenergetics and modulated STAT3 signaling. Oxygen consumption rate (OCR) was significantly increased, with no visible effect of 1,25(OH)2D3 treatment in A431∆PDIA3 cells. In the case of Extracellular Acidification Rate (ECAR), an increase was observed for glycolysis and glycolytic capacity parameters in the case of non-treated A431WT cells versus A431∆PDIA3 cells. The 1,25(OH)2D3 treatment had no significant effect on glycolytic parameters. Taken together, the presented results suggest that PDIA3 is strongly involved in the regulation of mitochondrial bioenergetics in cancerous cells and modulation of its response to 1,25(OH)2D3, possibly through STAT3.

PDIA3 modulates genomic response to 1,25-dihydroxyvitamin D3 in squamous cell carcinoma of the skin.

An active form of vitamin D3 (1,25-dihydroxyvitamin D3) acts through vitamin D receptor (VDR) initiating genomic response, but several studies described also non-genomic actions of 1,25-dihydroxyvitamin D3, implying the role of PDIA3 in the process. PDIA3 is a membrane-associated disulfide isomerase involved in disulfide bond formation, protein folding, and remodeling. Here, we used a transcriptome-based approach to identify changes in expression profiles in PDIA3-deficient squamous cell carcinoma line A431 after 1,25-dihydroxyvitamin D3 treatment. PDIA3 knockout led to changes in the expression of more than 2000 genes and modulated proliferation, cell cycle, and mobility of cells; suggesting an important regulatory role of PDIA3. PDIA3-deficient cells showed increased sensitivity to 1,25-dihydroxyvitamin D3, which led to decrease migration. 1,25-dihydroxyvitamin D3 treatment altered also genes expression profile of A431ΔPDIA3 in comparison to A431WT cells, indicating the existence of PDIA3-dependent genes. Interestingly, classic targets of VDR, including CAMP (Cathelicidin Antimicrobial Peptide), TRPV6 (Transient Receptor Potential Cation Channel Subfamily V Member 6), were regulated differently by 1,25-dihydroxyvitamin D3, in A431ΔPDIA3. Deletion of PDIA3 impaired 1,25-dihydroxyvitamin D3-response of genes, such as PTGS2, MMP12, and FOCAD, which were identified as PDIA3-dependent. Additionally, response to 1,25-dihydroxyvitamin D3 in cancerous A431 cells differed from immortalized HaCaT keratinocytes, used as non-cancerous control. Finally, silencing of PDIA3 and 1,25-dihydroxyvitamin D3, at least partially reverse the expression of cancer-related genes in A431 cells, thus targeting PDIA3 and use of 1,25-dihydroxyvitamin D3 could be considered in a prevention and therapy of the skin cancer. Taken together, PDIA3 has a strong impact on gene expression and physiology, including genomic response to 1,25-dihydroxyvitamin D3.

Size matters: the impact of nucleus size on results from spatial transcriptomics.

BACKGROUND: Visium Spatial Gene Expression (ST) is a method combining histological spatial information with transcriptomics profiles directly from tissue sections. The use of spatial information has made it possible to discover new modes of gene expression regulations. However, in the ST experiment, the nucleus size of cells may exceed the thickness of a tissue slice. This may, in turn, negatively affect comprehensive capturing the transcriptomics profile in a single slice, especially for tissues having large differences in the size of nuclei. METHODS: Here, we defined the effect of Consecutive Slices Data Integration (CSDI) on unveiling accurate spot clustering and deconvolution of spatial transcriptomic spots in human postmortem brains. By considering the histological information as reference, we assessed the improvement of unsupervised clustering and single nuclei RNA-seq and ST data integration before and after CSDI. RESULTS: Apart from the escalated number of defined clusters representing neuronal layers, the pattern of clusters in consecutive sections was concordant only after CSDI. Besides, the assigned cell labels to spots matches the histological pattern of tissue sections after CSDI. CONCLUSION: CSDI can be applied to investigate consecutive sections studied with ST in the human cerebral cortex, avoiding misinterpretation of spot clustering and annotation, increasing accuracy of cell recognition as well as improvement in uncovering the layers of grey matter in the human brain.

Assessment of the Potential Role of Selected Single Nucleotide Polymorphisms (SNPs) of Genes Related to the Functioning of Regulatory T Cells in the Pathogenesis of Psoriasis.

Recent studies have indicated a key role of the impaired suppressive capacity of regulatory T cells (Tregs) in psoriasis (PsO) pathogenesis. However, the genetic background of Treg dysfunctions remains unknown. The aim of this study was to evaluate the association of PsO development with selected single nucleotide polymorphisms (SNPs) of genes in which protein products play a significant role in the regulation of differentiation and function of Tregs. There were three study groups in our research and each consisted of different unrelated patients and controls: 192 PsO patients and 5605 healthy volunteers in the microarray genotyping group, 150 PsO patients and 173 controls in the ARMS-PCR method group, and 6 PsO patients and 6 healthy volunteers in the expression analysis group. The DNA microarrays analysis (283 SNPs of 57 genes) and ARMS-PCR method (8 SNPs in 7 genes) were used to determine the frequency of occurrence of SNPs in selected genes. The mRNA expression of selected genes was determined in skin samples. There were statistically significant differences in the allele frequencies of four SNPs in three genes (TNF, IL12RB2, and IL12B) between early-onset PsO patients and controls. The lowest p-value was observed for rs3093662 (TNF), and the G allele carriers had a 2.73 times higher risk of developing early-onset PsO. Moreover, the study revealed significant differences in the frequency of SNPs and their influence on PsO development between early- and late-onset PsO. Based on the ARMS-PCR method, the association between some polymorphisms of four genes (IL4, IL10, TGFB1, and STAT3) and the risk of developing PsO was noticed. Psoriatic lesions were characterized with a lower mRNA expression of FOXP3, CTLA4, and IL2, and a higher expression of TNF and IL1A in comparison with unaffected skin. In conclusion, the genetic background associated with properly functioning Tregs seems to play a significant role in PsO pathogenesis and could have diagnostic value.

Molecular Mechanisms of Neurogenic Inflammation of the Skin.

The skin, including the hypodermis, is the largest body organ and is in constant contact with the environment. Neurogenic inflammation is the result of the activity of nerve endings and mediators (neuropeptides secreted by nerve endings in the development of the inflammatory reaction in the skin), as well as interactions with other cells such as keratinocytes, Langerhans cells, endothelial cells and mast cells. The activation of TRPV-ion channels results in an increase in calcitonin gene-related peptide (CGRP) and substance P, induces the release of other pro-inflammatory mediators and contributes to the maintenance of cutaneous neurogenic inflammation (CNI) in diseases such as psoriasis, atopic dermatitis, prurigo and rosacea. Immune cells present in the skin (mononuclear cells, dendritic cells and mast cells) also express TRPV1, and their activation directly affects their function. The activation of TRPV1 channels mediates communication between sensory nerve endings and skin immune cells, increasing the release of inflammatory mediators (cytokines and neuropeptides). Understanding the molecular mechanisms underlying the generation, activation and modulation of neuropeptide and neurotransmitter receptors in cutaneous cells can aid in the development of effective treatments for inflammatory skin disorders.

Guidelines for Preventing and Treating Vitamin D Deficiency: A 2023 Update in Poland.

Introduction: All epidemiological studies suggest that vitamin D deficiency is prevalent among the Polish general population. Since vitamin D deficiency was shown to be among the risk factors for many diseases and for all-cause mortality, concern about this problem led us to update the previous Polish recommendations. Methods: After reviewing the epidemiological evidence, case-control studies and randomized control trials (RCTs), a Polish multidisciplinary group formulated questions on the recommendations for prophylaxis and treatment of vitamin D deficiency both for the general population and for the risk groups of patients. The scientific evidence of pleiotropic effects of vitamin D as well as the results of panelists' voting were reviewed and discussed. Thirty-four authors representing different areas of expertise prepared position statements. The consensus group, representing eight Polish/international medical societies and eight national specialist consultants, prepared the final Polish recommendations. Results: Based on networking discussions, the ranges of total serum 25-hydroxyvitamin D concentration indicating vitamin D deficiency [<20 ng/mL (<50 nmol/L)], suboptimal status [20-30 ng/mL (50-75 nmol/L)], and optimal concentration [30-50 ng/mL (75-125 nmol/L)] were confirmed. Practical guidelines for cholecalciferol (vitamin D3) as the first choice for prophylaxis and treatment of vitamin D deficiency were developed. Calcifediol dosing as the second choice for preventing and treating vitamin D deficiency was introduced. Conclusions: Improving the vitamin D status of the general population and treatment of risk groups of patients must be again announced as healthcare policy to reduce a risk of spectrum of diseases. This paper offers consensus statements on prophylaxis and treatment strategies for vitamin D deficiency in Poland.

VDR and PDIA3 Are Essential for Activation of Calcium Signaling and Membrane Response to 1,25(OH)2D3 in Squamous Cell Carcinoma Cells.

The genomic activity of 1,25(OH)2D3 is mediated by vitamin D receptor (VDR), whilst non-genomic is associated with protein disulfide isomerase family A member 3 (PDIA3). Interestingly, our recent studies documented that PDIA3 is also involved, directly or indirectly, in the modulation of genomic response to 1,25(OH)2D3. Moreover, PDIA3 was also shown to regulate cellular bioenergetics, possibly through the modulation of STAT signaling. Here, the role of VDR and PDIA3 proteins in membrane response to 1,25(OH)2D3 and calcium signaling was investigated in squamous cell carcinoma A431 cell line with or without the deletion of VDR and PDIA3 genes. Calcium influx was assayed by Fura-2AM or Fluo-4AM, while calcium-regulated element (NFAT) activation was measured using a dual luciferase assay. Further, the levels of proteins involved in membrane response to 1,25(OH)2D3 in A431 cell lines were analyzed via Western blot analysis. The deletion of either PDIA3 or VDR resulted in the decreased baseline levels of Ca2+ and its responsiveness to 1,25(OH)2D3; however, the effect was more pronounced in A431∆PDIA3. Furthermore, the knockout of either of these genes disrupted 1,25(OH)2D3-elicited membrane signaling. The data presented here indicated that the VDR is essential for the activation of calcium/calmodulin-dependent protein kinase II alpha (CAMK2A), while PDIA3 is required for 1,25(OH)2D3-induced calcium mobilization in A431 cells. Taken together, those results suggest that both VDR and PDIA3 are essential for non-genomic response to this powerful secosteroid.

Nongenomic Activities of Vitamin D.

Vitamin D shows a variety of pleiotropic activities which cannot be fully explained by the stimulation of classic pathway- and vitamin D receptor (VDR)-dependent transcriptional modulation. Thus, existence of rapid and nongenomic responses to vitamin D was suggested. An active form of vitamin D (calcitriol, 1,25(OH)2D3) is an essential regulator of calcium-phosphate homeostasis, and this process is tightly regulated by VDR genomic activity. However, it seems that early in evolution, the production of secosteroids (vitamin-D-like steroids) and their subsequent photodegradation served as a protective mechanism against ultraviolet radiation and oxidative stress. Consequently, direct cell-protective activities of vitamin D were proven. Furthermore, calcitriol triggers rapid calcium influx through epithelia and its uptake by a variety of cells. Subsequently, protein disulfide-isomerase A3 (PDIA3) was described as a membrane vitamin D receptor responsible for rapid nongenomic responses. Vitamin D was also found to stimulate a release of secondary massagers and modulate several intracellular processes-including cell cycle, proliferation, or immune responses-through wingless (WNT), sonic hedgehog (SSH), STAT1-3, or NF-kappaB pathways. Megalin and its coreceptor, cubilin, facilitate the import of vitamin D complex with vitamin-D-binding protein (DBP), and its involvement in rapid membrane responses was suggested. Vitamin D also directly and indirectly influences mitochondrial function, including fusion-fission, energy production, mitochondrial membrane potential, activity of ion channels, and apoptosis. Although mechanisms of the nongenomic responses to vitamin D are still not fully understood, in this review, their impact on physiology, pathology, and potential clinical applications will be discussed.

Current and Future Perspectives for Chimeric Antigen Receptor T Cells Development in Poland.

Chimeric antigen receptor T (CAR-T) cells are genetically modified autologous T cells that have revolutionized the treatment of relapsing and refractory haematological malignancies. In this review we present molecular pathways involved in the activation of CAR-T cells, describe in details the structures of receptors and the biological activity of CAR-T cells currently approved for clinical practice in the European Union, and explain the functional differences between them. Finally, we present the potential for the development of CAR-T cells in Poland, as well as indicate the possible directions of future research in this area, including novel modifications and applications of CAR-T cells and CAR-natural killer (NK) cells.

Vitamin D Signaling in Psoriasis: Pathogenesis and Therapy.

Psoriasis is a systemic, chronic, immune-mediated disease that affects approximately 2-3% of the world's population. The etiology and pathophysiology of psoriasis are still unknown, but the activation of the adaptive immune system with the main role of T-cells is key in psoriasis pathogenesis. The modulation of the local neuroendocrine system with the downregulation of pro-inflammatory and the upregulation of anti-inflammatory messengers represent a promising adjuvant treatment in psoriasis therapies. Vitamin D receptors and vitamin D-mediated signaling pathways function in the skin and are essential in maintaining the skin homeostasis. The active forms of vitamin D act as powerful immunomodulators of clinical response in psoriatic patients and represent the effective and safe adjuvant treatments for psoriasis, even when high doses of vitamin D are administered. The phototherapy of psoriasis, especially UVB-based, changes the serum level of 25(OH)D, but the correlation of 25(OH)D changes and psoriasis improvement need more clinical trials, since contradictory data have been published. Vitamin D derivatives can improve the efficacy of psoriasis phototherapy without inducing adverse side effects. The anti-psoriatic treatment could include non-calcemic CYP11A1-derived vitamin D hydroxyderivatives that would act on the VDR or as inverse agonists on RORs or activate alternative nuclear receptors including AhR and LXRs. In conclusion, vitamin D signaling can play an important role in the natural history of psoriasis. Selective targeting of proper nuclear receptors could represent potential treatment options in psoriasis.

Diet, Sun, Physical Activity and Vitamin D Status in Children with Inflammatory Bowel Disease.

In the course of inflammatory bowel disease (IBD) malabsorption may lead to a vitamin D deficiency and calcium-phosphate misbalance. However, the reports on the vitamin D status in children with IBD are few and ambiguous. Here, we are presenting complex analyses of multiple factors influencing 25OHD levels in IBD children (N = 62; Crohn's disease n = 34, ulcerative colitis n = 28, mean age 14.4 ± 3.01 years, F/M 23/39) and controls (n = 47, mean age 13.97 ± 2.57, F/M 23/24). Additionally, calcium-phosphate balance parameters and inflammatory markers were obtained. In children with IBD disease, activity and location were defined. Information about therapy, presence of fractures and abdominal surgery were obtained from medical records. All subjects were surveyed on the frequency and extent of exposure to sunlight (forearms, partially legs for at least 30 min a day), physical activity (at least 30 min a day) and diet (3 days diary was analyzed with the program DIETA 5). The mean 25OHD level was higher in IBD patients compared to controls (18.1 ng/mL vs. 15.5 ng/mL; p = 0.03). Only 9.7% of IBD patients and 4.25% of controls had the optimal vitamin D level (30-50 ng/mL). Despite the higher level of 25OHD, young IBD patients showed lower calcium levels in comparison to healthy controls. There was no correlation between the vitamin D level and disease activity or location of gastrointestinal tract lesions. Steroid therapy didn't have much influence on the vitamin D level while vitamin D was supplemented. Regular sun exposure was significantly more common in the control group compared to the IBD group. We found the highest concentration of vitamin D (24.55 ng/mL) with daily sun exposure. There was no significant correlation between the vitamin D level and frequency of physical activity. The analysis of dietary diaries showed low daily intake of vitamin D in both the IBD and the control group (79.63 vs. 85.14 IU/day). Pediatric patients, both IBD and healthy individuals, require regular monitoring of serum vitamin D level and its adequate supplementation.

Mitochondrial potassium channels: A novel calcitriol target.

BACKGROUND: Calcitriol (an active metabolite of vitamin D) modulates the expression of hundreds of human genes by activation of the vitamin D nuclear receptor (VDR). However, VDR-mediated transcriptional modulation does not fully explain various phenotypic effects of calcitriol. Recently a fast non-genomic response to vitamin D has been described, and it seems that mitochondria are one of the targets of calcitriol. These non-classical calcitriol targets open up a new area of research with potential clinical applications. The goal of our study was to ascertain whether calcitriol can modulate mitochondrial function through regulation of the potassium channels present in the inner mitochondrial membrane. METHODS: The effects of calcitriol on the potassium ion current were measured using the patch-clamp method modified for the inner mitochondrial membrane. Molecular docking experiments were conducted in the Autodock4 program. Additionally, changes in gene expression were investigated by qPCR, and transcription factor binding sites were analyzed in the CiiiDER program. RESULTS: For the first time, our results indicate that calcitriol directly affects the activity of the mitochondrial large-conductance Ca2+-regulated potassium channel (mitoBKCa) from the human astrocytoma (U-87 MG) cell line but not the mitochondrial calcium-independent two-pore domain potassium channel (mitoTASK-3) from human keratinocytes (HaCaT). The open probability of the mitoBKCa channel in high calcium conditions decreased after calcitriol treatment and the opposite effect was observed in low calcium conditions. Moreover, using the AutoDock4 program we predicted the binding poses of calcitriol to the calcium-bound BKCa channel and identified amino acids interacting with the calcitriol molecule. Additionally, we found that calcitriol influences the expression of genes encoding potassium channels. Such a dual, genomic and non-genomic action explains the pleiotropic activity of calcitriol. CONCLUSIONS: Calcitriol can regulate the mitochondrial large-conductance calcium-regulated potassium channel. Our data open a new chapter in the study of non-genomic responses to vitamin D with potential implications for mitochondrial bioenergetics and cytoprotective mechanisms.

Disturbed expression of vitamin D and retinoic acid-related orphan receptors α and γ and of megalin in inflammatory skin diseases.

The pathogenesis of inflammatory skin diseases is associated with the abnormal activity of keratinocytes and immune cells infiltrate. Vitamin D3 deficiency can correlate with the increased incidence, severity and duration of inflammatory skin disorders. The exact mechanism on how vitamin D3 influences inflammatory skin diseases still requires clarification. However, it can be associated with the disturbances in transmembrane glycoprotein-LRP2/megalin, which is implicated in vitamin D3 transport to the cell, and defects in vitamin D-signalling through the nuclear receptors. Therefore, by using immunohistochemistry, we analysed the expression of LRP2/megalin, VDR, RORα and RORγ in allergic contact dermatitis, lichen simplex chronicus, sarcoidosis and psoriasis in comparison with the normal skin. We observed decreased expression of LRP2/megalin in all inflammatory lesions in comparison with the normal skin. Significant differences were also noticed in VDR, RORα and RORγ levels between inflammatory lesions and normal skin. Our research indicates disturbed expression of LRP2/megalin, VDR, RORα and RORγ in inflammatory skin lesions in comparison with normal skin. Therefore, we suggest that changes in the activity of these proteins may play role in pathogenesis of inflammatory skin disorders. Furthermore, we suggest that LRP2/megalin, VDR, RORα and RORy may serve as targets in therapy of these diseases.

The Brain-Skin Axis in Psoriasis-Psychological, Psychiatric, Hormonal, and Dermatological Aspects.

Psoriasis is a chronic inflammatory skin disease with systemic manifestation, in which psychological factors play an important role. The etiology of psoriasis is complex and multifactorial, including genetic background and environmental factors such as emotional or physical stress. Psychological stress may also play a role in exacerbation of psoriasis, by dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, sympathetic-adrenal-medullary axis, peripheral nervous system, and immune system. Skin cells also express various neuropeptides and hormones in response to stress, including the fully functional analog of the HPA axis. The deterioration of psoriatic lesions is accompanied by increased production of inflammatory mediators, which could contribute to the imbalance of neurotransmitters and the development of symptoms of depression and anxiety. Therefore, deregulation of the crosstalk between endocrine, paracrine, and autocrine stress signaling pathways contributes to clinical manifestations of psoriasis, which requires multidisciplinary approaches.

The Effects of Vitamin D on the Expression of IL-33 and Its Receptor ST2 in Skin Cells; Potential Implication for Psoriasis.

Interleukin 33 (IL-33) belongs to the IL-1 family and is produced constitutively by epithelial and endothelial cells of various organs, such as the skin. It takes part in the maintenance of tissue homeostasis, repair, and immune response, including activation of Th2 lymphocytes. Its involvement in pathogenesis of several inflammatory diseases including psoriasis was also suggested, but this is not fully understood. The aim of the study was to investigate expression of IL-33 and its receptor, ST2, in psoriasis, and the effects of the active form of vitamin D (1,25(OH)2D3) on their expression in skin cells. Here we examined mRNA and protein profiles of IL-33 and ST2 in 18 psoriatic patients and healthy volunteers by qPCR and immunostaining techniques. Potential effects of 1,25(OH)2D3 and its receptor (VDR) on the expression of IL-33 and ST2 were tested in cultured keratinocytes, melanocytes, fibroblasts, and basal cell carcinoma cells. It was shown that 1,25(OH)2D3 effectively stimulated expression of IL-33 and its receptor ST2's mRNAs in a time-dependent manner, in keratinocytes and to the lesser extends in melanocytes, but not in fibroblasts. Furthermore, the effect of vitamin D on expression of IL-33 and ST2 was VDR-dependent. Finally, we demonstrated that the expression of mRNA for IL-33 was mainly elevated in the psoriatic skin but not in its margin. Interestingly, ST2 mRNA was downregulated in psoriatic lesion compared to both marginal tissue as well as healthy skin. Our data indicated that vitamin D can modulate IL-33 signaling, opening up new perspectives for our understanding of the mechanism of vitamin D action in psoriasis therapy.

Modulation of dermal equivalent of hypothalamus-pituitary-adrenal axis in mastocytosis.

INTRODUCTION: Mastocytosis is a rare disease characterized by abnormal growth and accumulation of tissue mast cells (MC) in one or more organ systems and is classified as being either cutaneous mastocytosis (CM) or systemic mastocytosis (SM). In the pioneer studies of Slominski's group, a fully functional hypothalamic-pituitary-adrenal axis equivalent has been discovered in various tissues, including skin. AIM: In the present study we investigated potential involvement of hypothalamus-pituitary-adrenal (HPA) cutaneous equivalent in ongoing mastocytosis. MATERIAL AND METHODS: The expression of HPA elements: CRH, UCN1, UCN2, UCN3, CRHR1, POMC, MC1R, MC2R and NR3C1 was assessed for their mRNA level in skin biopsies of adult patients with mastocytosis and healthy donors (n = 16 and 19, respectively), while CRH, UCN1, CRHR1, ACTH and MC1R were selected for immunostaining assay (n = 13 and 7, respectively). The expression of CRH receptor 1 (CRHR1) isomers was investigated by RT-PCR. The ELISA was used for detection of cortisol, CRH, UCN and ACTH in the serum. RESULTS: The decrease in the expression of HPA element of skin equivalent was observed on both mRNA and protein levels, however quantification of immunohistochemical data was impeded due to melanin in epidermis. Furthermore, we observed infiltration of dermis with HPA elements overexpressing mononuclear cells, which is in the agreement with an in vitro study showing a high expression of HPA elements by mast cells. CONCLUSIONS: Taken together, it was confirmed that the expression elements of HPA was modulated in mastocytosis, thus the potential involvement of general and local stress responses in its pathogenesis should be postulated and further investigated.

Discovery of novel 3-hydroxyandrosta-5,7-Diene-17-Carboxylic acid derivatives as anti-inflammatory bowel diseases (IBD) agents.

A series of steroidal compounds based on 3-hydroxyandrosta-5,7-diene-17-carboxylic acid core structure were designed, synthesized and bio-evaluated for their anti-inflammatory potency. Among them, compound 5c, 6f, and 6q effectively inhibited the production of nitric oxide (NO) in lipopolysaccharide (LPS) stimulated RAW 264.7 macrophages. They inhibited the expression of inducible NO synthase (iNOS) and prostaglandin synthase-2 (COX-2) at mRNA level. Compound 6q displayed inhibitory effects on both iNOS and COX-2 expression in a concentration-dependent manner. Furthermore, 6q was found to effectively decrease the mRNA and protein levels of interleukin 6 (IL-6). Mechanically, 6q could potently downregulate NF-κB signaling via suppression of the Akt/PI3K pathway. Moreover, 6q demonstrated high in vivo anti-inflammatory activities in a mouse colitis model induced by dextran sulfate sodium (DSS). Taken together, these data indicate that 6q represents a novel and promising anti-inflammatory bowel diseases (IBD) agent worthy of further investigation.