Ricardo Boland

Vitamin D: Beyond bone

In recent years, vitamin D has been received increased attention due to the resurgence of vitamin D deficiency and rickets in developed countries and the identification of extraskeletal effects of vitamin D, suggesting unexpected benefits of vitamin D in health and disease, beyond bone health. The possibility of extraskeletal effects of vitamin D was first noted with the discovery of the vitamin D receptor (VDR) in tissues and cells that are not involved in maintaining mineral homeostasis and bone health, including skin, placenta, pancreas, breast, prostate and colon cancer cells, and activated T cells. However, the biological significance of the expression of the VDR in different tissues is not fully understood, and the role of vitamin D in extraskeletal health has been a matter of debate. This report summarizes recent research on the roles for vitamin D in cancer, immunity and autoimmune diseases, cardiovascular and respiratory health, pregnancy, obesity, erythropoiesis, diabetes, muscle function, and aging.

Subcellular distribution of native estrogen receptor α and β isoforms in rabbit uterus and ovary

The association of estrogen receptors with non‐nuclear/cytoplasmic compartments in target tissues has been documented. However, limited information is available on the distribution of estrogen receptor isoforms, specially with regard to the newly described β isotype. The subcellular localization of estrogen receptor α and β isoforms was investigated in rabbit uterus and ovary. Native α and β subtypes were immunodetected using specific antibodies after subjecting the tissue to fractionation by differential centrifugation. The ovary expressed α and β estrogen receptors in predominant association to cytosolic components. However, in the uterus, a substantial proportion of the total estrogen binding capacity and coexpression of the two isoforms was detected in mitochondria and microsomes. The mitochondrial‐enriched subfraction represented an important source of 17β‐estradiol binding, where the steroid was recognized in a stereospecific and high affinity manner. The existence of mitochondrial and membrane estrogen binding sites correlated with the presence of estrogen receptor α but mainly with estrogen receptor β proteins. Using macromolecular 17β‐estradiol derivatives in Ligand Blot studies, we could confirm that both α and β isoforms were expressed as the major estrogen binding proteins in the uterus, while estrogen receptor α was clearly the dominant isoform in the ovary. Other low molecular weight estrogen receptor α‐like proteins were found to represent an independent subpopulation of uterine binding sites, expressed to a lesser extent. This differential cellular partitioning of estrogen receptor α and β forms may contribute to the known diversity of 17β‐estradiol effects in target organs. Both estrogen receptor α and β expression levels and cellular localization patterns among tissues, add complexity to the whole estrogen signaling system, in which membrane and mitochondrial events could also be implicated. J. Cell. Biochem. 82:467–479, 2001.

1,25(OH)2‐vitamin D3 induces translocation of the vitamin D receptor (VDR) to the plasma membrane in skeletal muscle cells

1,25‐dihydroxy‐vitamin D3 (1,25(OH)2D3), the hormonally active form of vitamin D3, acts through two different mechanisms. In addition to regulating gene expression via the specific intracellular vitamin D receptor (VDR), 1,25(OH)2D3 induces rapid, non‐transcriptional responses involving stimulation of transmembrane signal transduction pathways. The activation of second messengers supports the hypothesis that a membrane‐bound steroid receptor similar to those that mediate peptide hormone biology exists. Skeletal muscle is a target tissue for 1,25(OH)2D3. Avian embryonic skeletal muscle cells (myoblasts/myotubes) have been shown to respond both genomically and non‐genomically to the hormone. The present study provides evidence indicating that short‐term treatment (1–10 min) with 1,25(OH)2D3 induces translocation of the VDR from the nuclear to the microsomal fraction in chick myoblasts. This translocation is blocked by colchicine, genistein, or herbimycin, suggesting the involvement of microtubular transport and tyrosine kinase/s in the relocation of the receptor. By isolation of plasma membranes, it was demonstrated that the hormone increases the amounts of VDR specifically in this fraction. These results suggest that the nuclear VDR may be the receptor that mediates the non‐genomic effects of 1,25(OH)2D3 in chick myoblasts. J. Cell. Biochem. 86: 128–135, 2002.

Presence of a 1,25-dihydroxy-vitamin D3 receptor in chick skeletal muscle myoblasts

The presence of a specific receptor for 1,25-dihydroxy-vitamin D3 was investigated in myoblasts released from chick embryo skeletal muscle by trypsin and collagenase treatment. Density gradient analysis of the cytosol obtained from these muscle cell preparations showed that 1,25-dihydroxy-vitamin D3 binds specifically to a 3.7 S macromolecule. Scatchard analysis yielded an equilibrium dissociation constant of 2.46 x 10(-10) M and a Nmax of 74 fmol/mg of cytosol protein. The data is in agreement with previous evidence which indicates that the action of the vitamin D metabolite on muscle Ca uptake is mediated by de novo protein and RNA synthesis, and supports the concept that muscle is a target organ for 1,25-dihydroxy-vitamin D3.

17β-Estradiol abrogates apoptosis in murine skeletal muscle cells through estrogen receptors: role of the phosphatidylinositol 3-kinase/Akt pathway

Estrogens can regulate apoptosis in various cellular systems. The present study shows that 17beta-estradiol (E2), at physiological concentrations, abrogates DNA damage, poly (ADP-ribose) polymerase cleavage, and mitochondrial cytochrome c release induced by H2O2 or etoposide in mouse skeletal muscle C2C12 cells. This protective action, which involved PI3K/Akt activation and Bcl-2 associated death agonist (BAD) phosphorylation, was inhibited by antibodies against the estrogen receptor (ER) alpha or beta isoforms, or transfecting siRNA specific for each isoform. The inhibition of the antiapoptotic action of E2 at the mitochondrial level was more pronounced when ER-beta was immunoneutralized or suppressed by mRNA silencing, whereas transfection of C2C12 cells with either ER-alpha siRNA or ER-beta siRNA blocked the activation of Akt by E2, suggesting differential involvement of ER isoforms depending on the step of the apoptotic/survival pathway evaluated. These results indicate that E2 exerts antiapoptotic effects in skeletal muscle cells which are mediated by ER-beta and ER-alpha and involve the PI3K/Akt pathway.

Acute stimulation of intestinal cell calcium influx induced by 17β-estradiol via the cAMP messenger system

Recent studies have provided evidence for nuclear estrogen receptor-mediated calcium transport in intestinal mucosal cells. The possibility that, in addition, estrogens directly stimulate intestinal Ca2+ fluxes through second-messenger pathways was investigated. Exposure of enterocytes isolated from female rat duodenum to low physiological levels of 17 beta-estradiol (10(-11), 10(-10) and 10(-8) M) rapidly (1-10 min) increased (50-170%) cell 45Ca2+ influx. 17 alpha-Estradiol, dihydrotestosterone and progesterone were devoid of activity, suggesting specificity of the estrogen effect. Maximum responses induced by 17 beta-estradiol (5 min at 10(-10) M) could be abolished to a great extent (84%) by pretreating the cells with verapamil (10 microM) and nitrendipine (1 microM), involving the activation of voltage-dependent Ca2+ channels in the fast increase of rat duodenal calcium uptake by the hormone. Evidence was obtained indicating that the acute estrogen stimulation of enterocyte Ca2+ influx is mediated by the cyclic AMP/PKA pathway. 17 beta-Estradiol rapidly increased cAMP content of rat duodenal cells in parallel to the changes in Ca2+ uptake. In addition, forskolin, dibutyryl cAMP and Sp-cAMPS mimicked and Rp-cAMPS suppressed the prompt 17 beta-estradiol-induced stimulation of Ca2+ influx. These results are consistent with a direct action of estrogens in the enterocyte, presumably a non-genomic one, initiated on the cell surface and resulting in rapid activation of the cAMP pathway and Ca2+ channels, which may be relevant for regulation of intestinal calcium transport.

Characterization of membrane estrogen binding proteins from rabbit uterus

Estrogens exert fast non-genomic actions in their target tissues which may involve the participation of receptors located at the cell membrane. Studies were performed to identify and characterize membrane-associated 17beta-estradiol binding proteins in rabbit uterus. Specific and saturable [3H]17beta-estradiol binding sites of high affinity (Kd = 0.36 nM) were detected in uterine microsomes at higher concentration than in cytosol (370 +/- 98 vs. 270 +/- 87 fmol/mg protein, respectively). Various other steroid hormones, the stereoisomer 17alpha-estradiol and the antiestrogen tamoxifen were significantly less effective than 17beta-estradiol to compete with the radioactive ligand for binding to the membranes. The microsome binding sites were trypsin-sensitive and could be extracted to a great extent (80-90%) with 0.4/0.6 M KCl. Assays of the marker enzyme glucose-6-P dehydrogenase excluded membrane contamination with cytosolic soluble components. Immunoblot analysis of particulate and soluble fractions using monoclonal antibodies against the transactivation, heat shock protein recognition, and steroid binding domains of the nuclear estrogen receptor (ER; 67 kDa), revealed lower concentrations of the ER in membranes and the presence of five additional immunoreactive proteins of 57, 50, 32, 28, and 11 kDa which were absent in cytosol. Moreover, the antibody against the steroid binding domain was as effective as an inhibitor for cytosolic and membrane specific radioligand binding. Extraction of microsomes with the nondenaturing detergent CHAPS allowed a 2-fold enrichment of ER-like binding proteins as shown by antibody labeling and [3H]17beta-estradiol binding analysis. The results of this work are consistent with the existence of novel 17beta-estradiol membrane binding proteins structurally related to the intracellular ER. Future studies should investigate whether any of these proteins are involved in the primary events (e.g. receptor function) mediating nongenomic estrogen effects.

VDR activation of intracellular signaling pathways in skeletal muscle

The purpose of this article is to review the activation of signal transduction pathways in skeletal muscle cells by the hormone 1α,25(OH)(2)-vitamin D(3) [1α,25(OH)(2)D(3)], focusing on the role of the vitamin D receptor (VDR). The hormone induces fast, non transcriptional responses, involving stimulation of the transmembrane second messenger systems adenylyl cyclase/cAMP/PKA, PLC/DAG+IP(3)/PKC, Ca(2+) messenger system and MAPK cascades. Short treatment with 1α,25(OH)(2)D(3) induces reverse translocation of the VDR from the nucleus to plasma membranes. Accordingly, a complex is formed in the caveolae between the VDR and TRCP3, integral protein of capacitative Ca(2+) entry (CCE), suggesting an association between both proteins and a functional role of the VDR in 1α,25(OH)(2)D(3) activation of CCE. Stimulation of tyrosine phosphorylation cascades by 1α,25(OH)(2)D(3) have demonstrated the formation of complexes between Src and the VDR. Through these mechanisms, 1α,25(OH)(2)D(3) plays an important function in contractility and myogenesis.

1α,25-Dihydroxy-vitamin-D3-induced Store-operated Ca2+ Influx in Skeletal Muscle Cells MODULATION BY PHOSPHOLIPASE C, PROTEIN KINASE C, AND TYROSINE KINASES

In skeletal muscle cells the steroid hormone 1α,25-dihydroxy-vitamin-D3(1,25(OH)2D3) nongenomically promotes Ca2+ release from intracellular stores and cation influx through both l-type and store-operated Ca2+(SOC) channels. In the present work we evaluated the regulation and kinetics of the 1,25(OH)2D3-stimulated SOC influx in chick muscle cells. Stimulation with 10−9 m 1,25(OH)2D3 in Ca2+-free medium resulted in a rapid (40–60 s) but transient [Ca2+] i rise, which correlated with sterol-dependent inositol 1,4,5-trisphosphate production. The SOC influx stimulated by the hormone was insensitive to bothl-type channel antagonists and polyphosphoinositide-specific phospholipase C (PPI-PLC) inhibitors but was fully inhibitable by La3+ and Ni2+. PPI-PLC blockade prior to 1,25(OH)2D3 stimulation suppressed both the [Ca2+] i transient and the SOC influx. 1,25(OH)2D3-induced SOC entry was markedly increased after 3 min of treatment (30% above basal) and then rapidly reached a steady-state level. The sterol-stimulated SOC influx was prevented by protein kinase C and tyrosine kinase inhibitors but unaffected by blockade of the protein kinase A pathway. None of these inhibitors altered the thapsigargin-induced SOC entry, suggesting the operation of a signaling mechanism different from that for sterol-dependent SOC influx. The present results indicate that 1,25(OH)2D3-induced activation of PPI-PLC is upstream to Ca2+ influx through SOC channels and point for a role of both protein kinase C and tyrosine kinases but not protein kinase A in the regulation of the sterol-dependent SOCE pathway.

Activation of Src kinase in skeletal muscle cells by 1,25-(OH)2-vitamin D3 correlates with tyrosine phosphorylation of the vitamin D receptor (VDR) and VDR-Src interaction

The rapid effect of 1α,25(OH)2‐vitamin D3 [1α,25(OH)2D3] on tyrosine kinase Src and its relationship to the vitamin D receptor (VDR) was investigated to further characterize the hormone signaling mechanism in chick muscle cells. Exposure of cultured myotubes to 1α,25(OH)2D3 caused a time‐dependent increase in Src activity, which was evident at 1 min (one‐fold) and reached a maximum at 5 min (15‐fold). Immunoblotting with anti‐phosphotyrosine antibody of immunoprecipitated Src showed that the hormone decreased Src tyrosine phosphorylation state with maximal effects at 5 min. Using a database for protein consensus motifs we found a putative tyrosine phosphorylation site (amino acids 164–170: KTFDTTY) within the primary sequence of the chick VDR. When the myotube VDR was immunoprecipitated it appeared onto SDS‐PAGE gels as a single band of 58 kDa recognized by an anti‐phosphotyrosine antibody. Prior treatment of cells with 1α,25(OH)2D3 significantly increased tyrosine phosphorylation of the VDR (two‐ to three‐fold above basal levels). In agreement with Src being a SH2‐domain containing protein involved in recognition of tyrosine‐phosphorylated targets, immunoprecipitation with anti‐Src antibody under native conditions followed by blotting with anti‐VDR antibody, or using the antibodies in inverse order, showed that the VDR co‐precipitates with Src, thus indicating the existence of a VDR/Src complex. Stimulation with the cognate VDR ligand significantly increased formation of the complex with respect to basal conditions. These results altogether provide the first evidence to date for 1α,25(OH)2D3 activation involving Src association to tyrosine phosphorylated VDR. J. Cell. Biochem. 79:274–281, 2000.