Nonclassical Target Tissues Research Articles

Regulation of the expression of the gene for the parathyroid hormone-like protein

Vitamin D requires two metabolic conversions, 25-hydroxylation in the liver and 1α-hydroxylation in the kidney, before its hormonal form, 1,25-dihydroxyvitamin D [1,25-(OH)2D], can bind to the vitamin D receptor (VDR) to modulate gene transcription and regulate mineral ion homeostasis. The receptor and metabolic enzymes are expressed in many tissues, however, which has long suggested that the vitamin D hormone could act in an autocrine, paracrine, or intracrine fashion to affect the biology of non-classical target tissues. Strong support for this model has been obtained for Toll-like receptor-mediated innate immunity in macrophages, for example. The classical view is that vitamin D exerts its effects on bone indirectly via control of calcium and phosphate homeostasis, despite expression of cyp27b1, the 25-hydroxyvitamin D-1α-hydroxylase, and the VDR in osteoblasts and chondrocytes. Recent molecular genetic studies have revealed direct, but non-essential roles for 1,25-(OH)2D in growth plate chondrocytes. Specific inactivation of the VDR in collagen type II-expressing chondrocytes leads to reduced RANKL expression and delayed osteoclastogenesis, which causes a transient increase in bone volume at the primary spongiosa. Chondrocyte-specific VDR-ablated mice also show reduced circulating levels of FGF23 and thus elevated serum phosphate concentrations. The mechanisms remain to be completely determined but appear to involve a 1,25-(OH)2D-induced secreted factor from chondrocytes that affects FGF23 production by neighboring osteoblasts. The phenotype of additional mutant mice models, including chondrocyte-specific inactivation or overexpression of cyp27b1, is being analyzed to provide further support for these results that show autocrine and paracrine roles for 1,25-(OH)2D during endochondral bone development.

Cell differentiation and vitamin D

After the discovery of the effect of vitamin D on cell differentiation by Abe, Suda and their colleagues, it has become clear that many of the vitamin D actions are mediated by their effect on terminal differentiation of various cells. Similarly important are the findings that the vitamin D receptors are almost ubiquitously distributed in various tissues, and the active metabolite of vitamin D exerts its effects in almost all types of cells. Thus, the actions of vitamin D are not confined to their classical target tissues that regulate calcium metabolism, but also are extended to the regulation of the proliferation and differentiation of various cells such as immunoregulatory cells, epidermal cells and malignant tumor cells. These discoveries have created a new era in the field of vitamin D endocrinology, and led to the development of vitamin D analogues that exert their effects mostly in the nonclassical target tissues with little effects on calcium metabolism. Efforts are currently under way to apply these analogues to the treatment of various disturbances such as immunological disorders, skin lesions and malignant tumors. This review briefly summarizes the current understandings on the effect of vitamin D on cell differentiation and proliferation, and discusses the future prospects of the application of these effects on the treatment of various disorders.

Evidence for two classes of 1,25-dihydroxyvitamin D3 binding sites in classical vs. nonclassical target tissues

Possible differences in 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] binding sites in classical and nonclassical target tissues were tested by Scatchard analysis of [3H]1,25(OH)2D3 binding in parallel chromatin preparations of rat kidney vs. testis. Two distinct binding components were resolved in kidney (p less than 0.005). Moreover, the single binding site in testis exhibited a 10-fold lower Kd (p less than 0.05) than did the principal binding site in kidney (50 +/- 4 vs. 405 +/- 142 pM). Secondly, regulation of [3H]1,25(OH)2D3 binding sites also differed. 1,25(OH)2D3 injection resulted in increased 1,25(OH)2D3 binding (p less than 0.05) in kidney (92%) and intestine (415%), but not in testis, lung or heart. These results suggest that the principal 1,25(OH)2D3 binding sites in classical targets kidney and intestine may be intrinsically different from those in at least some nonclassical targets.

Vitamin D independence of small calcium-binding proteins in nonclassical target tissues

The presence and regulation of Ca-binding proteins (CaBPs) were investigated in newly identified 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] target tissues. 45Ca(2+)-blot analysis of proteins in normal rats yielded a 45Ca2+ band comigrating with authentic calmodulin. Additionally, a parvalbumin-like band (mol mass = 15.4 +/- 0.3 kDa) was prominent in prostate, and a strong unidentified 45Ca2+ band was always evident in the testis (mol mass = 23.5 +/- 0.7 kDa). Lung, bladder, and especially prostate demonstrated 45Ca2+ bands comigrating with the intestinal vitamin D-related CaBP (CaBP-D9K; mol mass = 10.9 +/- 0.5 kDa). Most tissues (including testis, heart, and lung) exhibited low levels of a 45Ca2+ band comigrating with the renal CaBP-D28K (mol mass = 28.3 +/- 0.4 kDa). Importantly, 45Ca2+ binding to all detectable CaBPs was unchanged in these four tissues in vitamin D-deficient rats, despite substantial downregulation of the intestinal CaBP-D9K and renal CaBP-D28K. Neither immunoblot analysis (rabbit anti-rat renal CaBP-D28K) nor Northern analysis (rat brain CaBP-D28K cDNA) provided evidence for coidentity of the 28-kDa 45Ca2+ band with the CaBP-D28K. Conversely, immunoblot analysis of lung, but not prostate, cytosol provided evidence for specific immunocross-reactivity to rabbit anti-rat intestinal CaBP-D9K. Immunoblot analysis of the 9-kDa CaBP in lung further confirmed its vitamin D independence. In conclusion, the vitamin D independence of the CaBPs in these putative new 1,25(OH)2D3 targets suggests the absence of an obligatory relationship between 1,25(OH)2D3 effects and CaBP induction therein.(ABSTRACT TRUNCATED AT 250 WORDS)

Extrarenal receptor-effector-mechanisms for aldosterone: The sequence of effects on the cellular electrolyte transport in human lymphocytes and their implications for disorders of the water and electrolyte balances

High affinity aldosterone binding sites have not only been described in the classic target tissues such as the renal tubules, but also in non-classic target tissues such as the hippocampus, mammary gland, endothelial cells and, recently, human mononuclear leukocytes. An in vitro effect of aldosterone on intracellular sodium, potassium and calcium concentrations and cell volume was shown in human mononuclear leukocytes. In the absence of aldosterone, the intracellular Na+, K+ and Ca2+ concentrations and the cell volume decreased significantly, but remained constant when aldosterone (1.4 nmol/l) was added to the incubation medium. These effects of aldosterone were blocked by the aldosterone antagonist canrenone (140 nmol/l). The sodium/proton exchanger of the cell membrane could be identified as the primary target of the aldosterone action, possibly non-genomically mediated through membrane receptors. The clinical significance of this model was underlined by the demonstration of absent or a decreased number of mineralocorticoid receptors and the lack of electrolyte response to aldosterone in human mononuclear leukocytes of patients with pseudohypoaldosteronism and aldosteronism. Additionally, an abnormal effector mechanism could be demonstrated in human mononuclear leukocytes from essential hypertensives. These studies are the first to demonstrate the significance of extrarenal, nonepithelial mineralocorticoid receptors and the related effector mechanism in different disorders of the water and electrolyte balance in man.

Mineralocorticoid effector mechanism in human mononuclear leukocytes

Mineralocorticoid receptors and mineralocorticoid effector mechanism were determined in mononuclear leukocytes (MNL) from normal subjects. The hierarchy of affinities of competitors for the receptor was similar to that described in other non-classical target tissues for aldosterone. In spite of the relative high affinity of cortisol for the receptor, these binding sites are occupied in vivo by aldosterone and play a mineralocorticoid effect in terms of electrolyte content of the cells. The effect of aldosterone is to prevent the loss of electrolytes due to incubation in medium alone and this action is reversed by addition of actinomycin D. In addition, the incubation of the MNL with aldosterone plus human αANP leads to complete block of the action of aldosterone alone. This effect is not mediated by binding of αANP to mineralocorticoid receptors but is probably related to a some postereceptorial effect of aldosterone at the level of plasma membrane. We conclude that the model of MNL is a good tool for studying mineralocorticoid receptors regulation and consequent effector mechanism in humans.