Characterization of the vitamin D endocrine system in human sebocytes in vitro

Sebocytes are sebum-producing cells that form the sebaceous glands. We investigated the role of sebocytes as target cells for vitamin D metabolites and the existence of an enzymatic machinery for the local synthesis and metabolism of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃, calcitriol], the biologically active vitamin D metabolite, in these cell types. Expression of vitamin D receptor (VDR), vitamin D-25-hydroxylase (25OHase), 25-hydroxyvitamin D-1α-hydroxylase (1αOHase), and 1,25-dihydroxyvitamin D-24-hydroxylase (24OHase) was detected in SZ95 sebocytes in vitro using real time quantitative polymerase chain reaction. Splice variants of 1αOHase were identified by nested touchdown polymerase chain reaction. We demonstrated that incubation of SZ95 sebocytes with 1,25(OH)₂D₃ resulted in a cell culture condition-, time-, and dose-dependent modulation of cell proliferation, cell cycle regulation, lipid content and interleukin-6/interleukin-8 secretion in vitro. RNA expression of VDR and 24OHase was upregulated along with vitamin D analogue treatment. Although several other splice variants of 1αOHase were detected, our findings indicate that the full length product represents the major 1αOHase gene product in SZ95 cells. In conclusion, SZ95 sebocytes express VDR and the enzymatic machinery to synthesize and metabolize biologically active vitamin D analogues. Sebocytes represent target cells for biologically active metabolites. Our findings indicate that the vitamin D endocrine system is of high importance for sebocyte function and physiology. We conclude that sebaceous glands represent potential targets for therapy with vitamin D analogues or for pharmacological modulation of 1,25(OH)₂D₃ synthesis/metabolism.     

Proteasome dysfunction in Drosophila signals to an Nrf2‐dependent regulatory circuit aiming to restore proteostasis and prevent premature aging

The ubiquitin–proteasome system is central to the regulation of cellular proteostasis. Nevertheless, the impact of in vivo proteasome dysfunction on the proteostasis networks and the aging processes remains poorly understood. We found that RNAi‐mediated knockdown of 20S proteasome subunits in Drosophila melanogaster resulted in larval lethality. We therefore studied the molecular effects of proteasome dysfunction in adult flies by developing a model of dose‐dependent pharmacological proteasome inhibition. Impaired proteasome function promoted several ‘old‐age’ phenotypes and markedly reduced flies' lifespan. In young somatic tissues and in gonads of all ages, loss of proteasome activity induced higher expression levels and assembly rates of proteasome subunits. Proteasome dysfunction was signaled to the proteostasis network by reactive oxygen species that originated from malfunctioning mitochondria and triggered an Nrf2‐dependent upregulation of the proteasome subunits. RNAi‐mediated Nrf2 knockdown reduced proteasome activities, flies' resistance to stress, as well as longevity. Conversely, inducible activation of Nrf2 in transgenic flies upregulated basal proteasome expression and activity independently of age and conferred resistance to proteotoxic stress. Interestingly, prolonged Nrf2 overexpression reduced longevity, indicating that excessive activation of the proteostasis pathways can be detrimental. Our in vivo studies add new knowledge on the proteotoxic stress‐related regulation of the proteostasis networks in higher metazoans. Proteasome dysfunction triggers the activation of an Nrf2‐dependent tissue‐ and age‐specific regulatory circuit aiming to adjust the cellular proteasome activity according to temporal and/or spatial proteolytic demands. Prolonged deregulation of this proteostasis circuit accelerates aging.     

Identification of negative and positive regulatory elements in the human renin gene

Renin gene expression is tissue-specific and under complex hormonal control. To investigate which DNA elements are involved in the control of human renin gene expression, we performed transient DNA transfer experiments with renin-chloramphenicol acetyltransferase fusions. We have mapped a complex arrangement of positive and negative control sequences in the 5' flanking region of the human renin gene. One positive control element is active in either orientation and defines a renin gene enhancer. The negative element is also active in either orientation and defines a renin gene silencer. Mapping in the same region as the silencer is a cAMP-responsive element, a sequence conserved in mouse, rat, and human renin genes.     

Characterization of a cell-type-restricted negative regulatory activity of the human granulocyte-macrophage colony-stimulating factor gene.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the proliferation and maturation of normal myeloid progenitor cells and can also stimulate the growth of acute myelogenous leukemia (AML) blasts. GM-CSF is not normally produced by resting cells but is expressed by a variety of activated cells including T lymphocytes, macrophages, and certain cytokine-stimulated fibroblasts and endothelial cells. Production of GM-CSF by cultured AML cells has been demonstrated, and GM-CSF expression by normal myeloid progenitors has been postulated to play a role in myelopoiesis. We have investigated the regulation of expression of GM-CSF in AML cell lines, and our results demonstrate the presence of a strong constitutive promoter element contained within 53 bp upstream of the cap site. We have also identified a negative regulatory element located immediately upstream of the positive regulatory element (within 69 bp of the cap site) that is active in AML cell lines but not T cells or K562 CML cells. Competition transfection and mobility shift studies demonstrate that this activity correlates with binding of a 45-kDa protein.     

Klotho lacks a vitamin D independent physiological role in glucose homeostasis, bone turnover, and steady-state PTH secretion in vivo

Apart from its function as co-receptor for fibroblast growth factor-23 (FGF23), Klotho is thought to regulate insulin signaling, intracellular oxidative stress, and parathyroid hormone (PTH) secretion in an FGF23 independent fashion. Here, we crossed Klotho deficient (Kl^−/−) mice with vitamin D receptor (VDR) mutant mice to examine further vitamin D independent functions of Klotho. All mice were fed a rescue diet enriched with calcium, phosphorus, and lactose to prevent hyperparathyroidism in VDR mutants, and were killed at 4 weeks of age after double fluorochrome labeling. Kl^−/− mice displayed hypercalcemia, hyperphosphatemia, dwarfism, organ atrophy, azotemia, pulmonary emphysema, and osteomalacia. In addition, glucose and insulin tolerance tests revealed hypoglycemia and profoundly increased peripheral insulin sensitivity in Kl^−/− mice. Compound mutants were normocalcemic and normophosphatemic, did not show premature aging or organ atrophy, and were phenocopies of VDR mutant mice in terms of body weight, bone mineral density, bone metabolism, serum calcium, serum phosphate, serum PTH, gene expression in parathyroid glands, as well as urinary calcium and phosphate excretion. Furthermore, ablation of vitamin D signaling in double mutants completely normalized glucose and insulin tolerance, indicating that Klotho has no vitamin D independent effects on insulin signaling. Histomorphometry of pancreas islets showed similar beta cell volume per body weight in all groups of animals. In conclusion, our findings cast doubt on a physiologically relevant vitamin D and Fgf23 independent function of Klotho in the regulation of glucose metabolism, bone turnover, and steady-state PTH secretion in vivo.     

Association of VDR-gene variants with factors related to the metabolic syndrome,type 2 diabetes and vitamin D deficiency

The prevalence of metabolic syndrome (MetS) is rising alarmingly in the Saudi Arabian population. This study was conducted to assess the association between vitamin D receptor (VDR) polymorphisms and genetic susceptibility to components of the metabolic syndrome, type 2 diabetes mellitus (T2DM), and vitamin D deficiency in the Saudi Arabian population. Five-hundred-seventy Saudi individuals (285 MetS and 285 controls) were enrolled in this cross-sectional study. TaqI, BsmI, ApaI and FokI single nucleotide polymorphisms (SNPs) of the VDR gene were genotyped. The CT genotype and allele T of BsmI were associated with lower HDL-C levels [OR 0.60 (0.37, 0.96), p = 0.03] and obesity [OR 1.4 (1.0, 1.90), p = 0.04], respectively. The CT genotype and the dominant model CT + TT of BsmI were associated with increased risk of diabetes [OR 1.7 (1.2, 2.4), p = 0.007], and [OR 1.5 (1.1, 2.2), p = 0.01], respectively. On the contrary, the CT and CT + CC genotypes of FokI exhibited an association with a reduced risk of diabetes [OR 0.70 (0.49, 0.99), p = 0.05] and [OR 0.67 (0.48, 0.94), p = 0.02], respectively. The allele C of FokI was associated with lower risk of developing T2DM [OR 0.73 (0.56, 0.95), p = 0.02]. The prevalence of vitamin D deficiency was lower in subjects with the AC genotype of ApaI [OR, 0.34 (0.14, 0.80), p = 0.01]. Components of the MetS such as obesity, low HDL and T2DM were associated with the VDR gene. FokI and BsmI have protective and facilitative effects on the risk for T2DM, while the ApaI genotype was associated with reduced vitamin D deficiency.     

Multiple forms of phospholipase D in plants: the gene family, catalytic and regulatory properties, and cellular functions

Multiple Phospholipase D (PLD) genes have been identified in plants and encode isoforms with distinct regulatory and catalytic properties. Elucidation of the genetic and biochemical heterogeneity has provided important clues as to the regulation and function of this family of enzymes. Polyphosphoinositides, Ca(2+), and G-proteins are possible cellular regulators for PLD activation. PLD-mediated hydrolysis of membrane lipids increases in response to various stresses. Recent studies suggest that PLD plays a role in the signaling and production of hormones involved in plant stress responses.