Ultrastructural characterization of vitamin D receptors and metabolizing enzymes in the lipid droplets of the fatty liver in rat.

Vitamin D is a steroid hormone with numerous actions in the organism. There are strong evidences that relate vitamin D deficiency with liver lipid metabolism disturbances, but the mechanism of this action is still unknown. In our previous work we postulated the localization and accumulation of vitamin D receptor (VDR) in membrane of the lipid droplets (LDs) in hepatocytes. In this study, we applied the transmission electron microscopy (TEM) to confirm this hypothesis by using a long-term (6 months) high sucrose intake rat model that was previously found to be appropriate for research of the hepatic lipid accumulation. In addition to the VDR, we also found key vitamin D metabolizing enzymes, 1α-hydroxylase and CYP 24 associated with the membrane of the LDs. A light-microscopy data revealed significant increase in expression of VDR and CYP 24 in liver of high-sucrose treated rats, in comparison to controlones. According to the best of our knowledge, this is a first study confirming the presence of the VDR in the membrane of the LDs in general and also in particular in LDs of the hepatocytes that were accumulated as a consequence of the prolonged high sucrose intake. Moreover, we found association of main vitamin D metabolizing enzymes with LD membrane. These results provide a new insight in the possible relation of vitamin D signalling system with LD morphology and function and with the lipid metabolism in general.

The role of receptor topology in the vitamin D3 uptake and Ca(2+) response systems.

The steroid hormone, vitamin D3, regulates gene transcription via at least two receptors and initiates putative rapid response systems at the plasma membrane. The vitamin D receptor (VDR) binds vitamin D3 and a second receptor, importin-4, imports the VDR-vitamin D3 complex into the nucleus via nuclear pores. Here we present evidence that the Homo sapiens VDR homodimer contains two transmembrane (TM) helices ((327)E - D(342)), two TM "half-helix" ((264)K N(276)), one or more large channels, and 16 cholesterol binding (CRAC/CARC) domains. The importin-4 monomer exhibits 3 pore-lining regions ((226)E - L(251); (768)V - G(783); (876)S - A(891)) and 16 CRAC/CARC domains. The MEMSAT algorithm indicates that VDR and importin-4 may not be restricted to cytoplasm and nucleus. VDR homodimer TM helix-topology predicts insertion into the plasma membrane, with two 84 residue C-terminal regions being extracellular. Similarly, MEMSAT predicts importin-4 insertion into the plasma membrane with 226 residue extracellular N-terminal regions and 96 residue C-terminal extracellular loops; with the pore-lining regions contributing gated Ca(2+) channels. The PoreWalker algorithm indicates that, of the 427 residues in each VDR monomer, 91 line the largest channel, including two vitamin D3 binding sites and residues from both the TM helix and "half-helix". Cholesterol-binding domains also extend into the channel within the ligand binding region. Programmed changes in bound cholesterol may regulate both membrane Ca(2+) response systems and vitamin D3 uptake as well as receptor internalization by the endomembrane system culminating in uptake of the vitamin D3-VDR-importin-4 complex into the nucleus.

Aspectos toxicológico, clínico-patológico e ultraestrutural das intoxicações iatrogênica e experimental por vitamina D em coelhos / Toxicological, clinic-pathological and ultrastructural aspects of iatrogenic and experimental poisoning by vitamin D in rabbits

São descritos aspectos toxicológicos, clínico-patológicos e ultraestruturais de coelhos intoxicados iatrogênica e experimentalmente por vitamina D por via subcutânea. Clinicamente, os animais evidenciaram sinais de insuficiência cardiovascular como ascite e edema pulmonar, hiporexia, anorexia, diarréia mucosa, emagrecimento e apatia. As clássicas alterações de mineralização e, por vezes, osseificação, do sistema cardiovascular, bem como as alterações de rins, pulmões, estômago, entre outros órgãos, foram reproduzidas com administrações subcutâneas de solução oleosa de colecalciferol (vitamina D3 não-ativada).

Toxicological, clinic-pathological and ultrastructural aspects of iatrogenic and experimental subcutaneous poisoning in rabbits by vitamin D are described. Clinically the animals showed signs of cardiovascular insufficiency, as ascite and lung edema, hyporexia, anorexia, mucous diarrhoea, loss of weight and apathy. The classical alterations of minera-lization and, occasionally, ossification of the cardiovascular system, as well the lesions of kidneys, lungs, stomach, among other organs, were reproduced by the subcutaneous administration of an oily solution of cholecalciferol (non-activated vitamin D3).

Ligand unbinding pathways from the vitamin D receptor studied by molecular dynamics simulations.

Molecular dynamics simulation techniques have been used to study the unbinding pathways of 1alpha,25-dihydroxyvitamin D(3) from the ligand-binding pocket of the vitamin D receptor (VDR). The pathways observed in a large number of relatively short (< 200 ps) random acceleration molecular dynamics (RAMD) trajectories were found to be in fair agreement, both in terms of pathway locations and deduced relative preferences, compared to targeted molecular dynamics (TMD) and streered molecular dynamics simulations (SMD). However, the high-velocity ligand expulsions of RAMD tend to favor straight expulsion trajectories and the observed relative frequencies of different pathways were biased towards the probability of entering a particular exit channel. Simulations indicated that for VDR the unbinding pathway between the H1-H2 loop and the beta-sheet between H5 and H6 is more favorable than the pathway located between the H1-H2 loop and H3. The latter pathway has been suggested to be the most likely unbinding path for thyroid hormone receptors (TRs) and a likely path for retinoic acid receptor. Ligand entry/exit through these two pathways would not require displacement of H12 from its agonistic position. Differences in the packing of the H1, H2, H3 and beta-sheet region explain the changed relative preference of the two unbinding pathways in VDR and TRs. Based on the crystal structures of the ligand binding domains of class 2 nuclear receptors, whose members are VDR and TRs, this receptor class can be divided in two groups according to the packing of the H1, H2, H3 and beta-sheet region.

Immunoreactivity of six monoclonal antibodies directed against 1,25-dihydroxyvitamin-D3 receptors in human skin.

Using confocal laser scanning microscopy, we tested the suitability of five monoclonal mouse antibodies (IVA7E7, IVB12G12, IVG9C11, VD2F12, and VIIID8C12) that had been raised against different domains of the porcine intestinal 1,25-dihydroxyvitamin-D3 receptor (VDR), for the immunohistological detection of VDR in human skin. The VDR immunoreactivity of these antibodies was compared with the well-characterized VDR-staining pattern of the mouse monoclonal antibody 9A7gamma raised against chick intestinal VDR. All six antibodies revealed strong nuclear and qualitatively similar immunoreactivity in all cell layers of the viable epidermis. Our data demonstrate that the five mouse monoclonal antibodies are suitable for immunohistochemical detection of VDR in frozen sections. These antibodies show comparable staining patterns in human skin even though they had been raised against different functional domains of the 1,25-dihydroxyvitamin-D3 receptor.

Biologic effects of topical calcipotriol (MC 903) treatment in psoriatic skin.

BACKGROUND: The biologically active vitamin D analog calcipotriol is effective and safe in the topical treatment of psoriasis, but its exact mechanism of action is unknown. OBJECTIVE: We investigated expression of 1,25-dihydroxyvitamin D3 receptors, markers for inflammation (CD1a, CD4, CD8, CD11b, CD15; NAP-1/interleukin-8; 55 kd tumor necrosis factor-receptor; intercellular adhesion molecule-1; HLA-DR), proliferation (proliferating cell nuclear antigen, Ki-67), and differentiation (transglutaminase K; involucrin; cytokeratin 16) in psoriatic skin during topical calcipotriol treatment. METHODS: For immunohistochemical staining we used the labeled avidin-biotin technique on cryostat-cut sections. RESULTS: We found a significant increase of 1,25-dihydroxyvitamin D3 receptor expression in epidermal basal keratinocytes of lesional psoriatic skin during calcipotriol treatment. In all patients analyzed, effects on proliferation and differentiation of epidermal keratinocytes were stronger than effects on dermal inflammation. Effects on inflammation were more pronounced in the epidermal than in the dermal compartment. CONCLUSION: Our findings indicate that analogs of 1,25-dihydroxyvitamin D3 upregulate their corresponding receptor in human keratinocytes in vivo. This mechanism may be important in the therapeutic efficacy of vitamin D analogs in psoriasis. The differential therapeutic effects in the epidermal and dermal skin compartments may be due to a reduced bioavailability of calcipotriol in the dermal compartment.

Conformational change and enhanced stabilization of the vitamin D receptor by the 1,25-dihydroxyvitamin D3 analog KH1060.

The 1,25-dihydroxyvitamin D3 [1,25-(OH)2vitamin D3] analog KH1060 exerts very potent effects on cell proliferation and cell differentiation via the vitamin D receptor (VDR). However, the activities of KH1060 are not associated with an increased affinity for the VDR. We now show that increased stabilization of the VDR-KH1060 complex could be an explanation for its high potencies. VDR half-life studies performed with cycloheximide-translational blocked rat osteoblast-like ROS 17/2.8 cells demonstrated that, in the absence of ligand, VDR levels rapidly decreased. After 2 hr, less than 10% of the initial VDR level could be measured. In the presence of 1,25-(OH)2vitamin D3, the VDR half-life was 15 hr. After 24 hr. less than 20% of the initial VDR content was detectable, whereas, at this time-point, when the cells were incubated with KH1060 80% of the VDR was still present. Differences in 1,25-(OH)2vitamin D3- and KH1060-induced conformational changes of the VDR could underlie the increased VDR stability. As assessed by limited proteolytic digestion analysis, both 1,25-(OH)2vitamin D3 and KH1060 caused a specific conformational change of the VDR. Compared with 1,25-(OH)2vitamin D3, KH1060 induced a conformational change that led to a far more dramatic protection of the VDR against proteolytic degradation. In conclusion, the altered VDR stability and the possibly underlying change in VDR conformation caused by KH1060 could be an explanation for its enhanced bioactivity.