GLIS3 expression in the thyroid gland in relation to TSH signaling and regulation of gene expression.

Loss of GLI-Similar 3 (GLIS3) function in mice and humans causes congenital hypothyroidism (CH). In this study, we demonstrate that GLIS3 protein is first detectable at E15.5 of murine thyroid development, a time at which GLIS3 target genes, such as Slc5a5 (Nis), become expressed. This, together with observations showing that ubiquitous Glis3KO mice do not display major changes in prenatal thyroid gland morphology, indicated that CH in Glis3KO mice is due to dyshormonogenesis rather than thyroid dysgenesis. Analysis of GLIS3 in postnatal thyroid suggested a link between GLIS3 protein expression and blood TSH levels. This was supported by data showing that treatment with TSH, cAMP, or adenylyl cyclase activators or expression of constitutively active PKA enhanced GLIS3 protein stability and transcriptional activity, indicating that GLIS3 activity is regulated at least in part by TSH/TSHR-mediated activation of PKA. The TSH-dependent increase in GLIS3 transcriptional activity would be critical for the induction of GLIS3 target gene expression, including several thyroid hormone (TH) biosynthetic genes, in thyroid follicular cells of mice fed a low iodine diet (LID) when blood TSH levels are highly elevated. Like TH biosynthetic genes, the expression of cell cycle genes is suppressed in ubiquitous Glis3KO mice fed a LID; however, in thyroid-specific Glis3 knockout mice, the expression of cell cycle genes was not repressed, in contrast to TH biosynthetic genes. This indicated that the inhibition of cell cycle genes in ubiquitous Glis3KO mice is dependent on changes in gene expression in GLIS3 target tissues other than the thyroid.

Circadian regulation of Ca V 1.2 expression by RORα in the mouse heart.

Background: In addition to show autonomous beating rhythmicity, the physiological functions of the heart present daily periodic oscillations. Notably the ventricular repolarization itself varies throughout the circadian cycle which was mainly related to the periodic expression of K + channels. However, the involvement of the L-type Ca 2+ channel (Ca V 1.2 encoded by Cacna1c gene) in these circadian variations remains elusive. Methods: We used a transgenic mouse model (PCa-luc) that expresses the luciferase reporter under the control of the cardiac Cacna1c promoter and analyzed promoter activity by bioluminescent imaging, qPCR, immunoblot, Chromatin immunoprecipitation assay (ChIP) and Ca V 1.2 activity. Results: Under normal 12:12h light-dark cycle, we observed in vivo a biphasic diurnal variation of promoter activities peaking at 9 and 19.5 Zeitgeber time (ZT). This was associated with a periodicity of Cacna1c mRNA levels preceding 24-h oscillations of Ca V 1.2 protein levels in ventricle (with a 1.5 h phase shift) but not in atrial heart tissues. The periodicity of promoter activities and Ca V 1.2 proteins, which correlated with biphasic oscillations of L-type Ca 2+ current conductance, persisted in isolated ventricular cardiomyocytes from PCa-Luc mice over the course of the 24-h cycle, suggesting an endogenous cardiac circadian regulation. Comparison of 24-h temporal patterns of clock gene expressions in ventricles and atrial tissues of the same mice revealed conserved circadian oscillations of the core clock genes except for the retinoid-related orphan receptor α gene (RORα), which remained constant throughout the course of a day in atrial tissues. In vitro we found that RORα is recruited to two specific regions on the Cacna1c promoter and that incubation with specific RORα inhibitor disrupted 24-h oscillations of ventricular promoter activities and Ca V 1.2 protein levels. Similar results were observed for pore forming subunits of the K + transient outward currents, K V 4.2 and K V 4.3. Conclusions: These findings raise the possibility that the RORα-dependent rhythmic regulation of cardiac Ca V 1.2 and K V 4.2/4.3 throughout the daily cycle may play an important role in physiopathology of heart function.

Biological Effects of CYP11A1-Derived Vitamin D and Lumisterol Metabolites in the Skin.

Novel pathways of vitamin D3, lumisterol 3 (L3), and tachysterol 3 (T3) activation have been discovered, initiated by CYP11A1 and/or CYP27A1 in the case of L3 and T3. The resulting hydroxymetabolites enhance protection of skin against DNA damage and oxidative stress; stimulate keratinocyte differentiation; exert anti-inflammatory, antifibrogenic, and anticancer activities; and inhibit cell proliferation in a structure-dependent manner. They act on nuclear receptors, including vitamin D receptor, aryl hydrocarbon receptor, LXRα/ß, RAR-related orphan receptor α/γ, and peroxisome proliferator-activated receptor-γ, with selectivity defined by their core structure and distribution of hydroxyl groups. They can activate NRF2 and p53 and inhibit NF-κB, IL-17, Shh, and Wnt/ß-catenin signaling. Thus, they protect skin integrity and physiology.

Glis2 is an early effector of polycystin signaling and a target for therapy in polycystic kidney disease.

Mouse models of autosomal dominant polycystic kidney disease (ADPKD) show that intact primary cilia are required for cyst growth following the inactivation of polycystin-1. The signaling pathways underlying this process, termed cilia-dependent cyst activation (CDCA), remain unknown. Using translating ribosome affinity purification RNASeq on mouse kidneys with polycystin-1 and cilia inactivation before cyst formation, we identify the differential 'CDCA pattern' translatome specifically dysregulated in kidney tubule cells destined to form cysts. From this, Glis2 emerges as a candidate functional effector of polycystin signaling and CDCA. In vitro changes in Glis2 expression mirror the polycystin- and cilia-dependent changes observed in kidney tissue, validating Glis2 as a cell culture-based indicator of polycystin function related to cyst formation. Inactivation of Glis2 suppresses polycystic kidney disease in mouse models of ADPKD, and pharmacological targeting of Glis2 with antisense oligonucleotides slows disease progression. Glis2 transcript and protein is a functional target of CDCA and a potential therapeutic target for treating ADPKD.

Novel Vitamin D3 Hydroxymetabolites Require Involvement of the Vitamin D Receptor or Retinoic Acid-Related Orphan Receptors for Their Antifibrogenic Activities in Human Fibroblasts.

We investigated multiple signaling pathways activated by CYP11A1-derived vitamin D3 hydroxymetabolites in human skin fibroblasts by assessing the actions of these molecules on their cognate receptors and by investigating the role of CYP27B1 in their biological activities. The actions of 20(OH)D3, 20,23(OH)2D3, 1,20(OH)2D3 and 1,20,23(OH)3D3 were compared to those of classical 1,25(OH)2D3. This was undertaken using wild type (WT) fibroblasts, as well as cells with VDR, RORs, or CYP27B1 genes knocked down with siRNA. Vitamin D3 hydroxymetabolites had an inhibitory effect on the proliferation of WT cells, but this effect was abrogated in cells with silenced VDR or RORs. The collagen expression by WT cells was reduced upon secosteroid treatment. This effect was reversed in cells where VDR or RORs were knocked down where the inhibition of collagen production and the expression of anti-fibrotic genes in response to the hydroxymetabolites was abrogated, along with ablation of their anti-inflammatory action. The knockdown of CYP27B1 did not change the effect of either 20(OH)D3 or 20,23(OH)2D3, indicating that their actions are independent of 1α-hydroxylation. In conclusion, the expression of the VDR and/or RORα/γ receptors in fibroblasts is necessary for the inhibition of both the proliferation and fibrogenic activity of hydroxymetabolites of vitamin D3, while CYP27B1 is not required.

Malignant Melanoma: An Overview, New Perspectives, and Vitamin D Signaling.

Melanoma, originating through malignant transformation of melanin-producing melanocytes, is a formidable malignancy, characterized by local invasiveness, recurrence, early metastasis, resistance to therapy, and a high mortality rate. This review discusses etiologic and risk factors for melanoma, diagnostic and prognostic tools, including recent advances in molecular biology, omics, and bioinformatics, and provides an overview of its therapy. Since the incidence of melanoma is rising and mortality remains unacceptably high, we discuss its inherent properties, including melanogenesis, that make this disease resilient to treatment and propose to use AI to solve the above complex and multidimensional problems. We provide an overview on vitamin D and its anticancerogenic properties, and report recent advances in this field that can provide solutions for the prevention and/or therapy of melanoma. Experimental papers and clinicopathological studies on the role of vitamin D status and signaling pathways initiated by its active metabolites in melanoma prognosis and therapy are reviewed. We conclude that vitamin D signaling, defined by specific nuclear receptors and selective activation by specific vitamin D hydroxyderivatives, can provide a benefit for new or existing therapeutic approaches. We propose to target vitamin D signaling with the use of computational biology and AI tools to provide a solution to the melanoma problem.