G.J.C.M. van den Bemd

Evidence for Involvement of 17β-Estradiol in Intestinal Calcium Absorption Independent of 1,25-Dihydroxyvitamin D3 Level in the Rat

The sex steroid 17β‐estradiol (17β‐E2) has a broad range of actions, including effects on calcium and bone metabolism. This study with 3‐month‐old Brown Norway rats was designed to investigate the role of 17β‐E2 in the regulation of calcium homeostasis. Rats were divided in four groups, sham‐operated, ovariectomized (OVX), and OVX supplemented with either a 0.025‐mg or 0.05‐mg 17β‐E2 pellet implanted subcutaneously. After 4 weeks, in none of the groups was serum calcium, phosphate, or parathyroid hormone altered compared with the sham group, while only in the OVX rats was a significant reduction in urinary calcium found. Bone mineral density and osteocalcin were modified, as can be expected after OVX and 17β‐E2 supplementation. OVX resulted in a nonsignificant increase in serum 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3). Supplementation with either one of the 17β‐E2 dosages resulted in an 80% reduction of 1,25(OH)2D3 and only a 20% reduction in 25‐hydroxyvitamin D3 levels. OVX, as well as supplementation with 17β‐E2, did not affect serum levels of vitamin D binding protein. As a consequence, the estimated free 1,25(OH)2D3 levels were also significantly decreased in the 17β‐E2‐supplemented group compared with the sham and OVX groups. Next, the consequences for intestinal calcium absorption were analyzed by the in situ intestinal loop technique. Although the 1,25(OH)2D3 serum level was increased, OVX resulted in a significant decrease in intestinal calcium absorption in the duodenum. Despite the strongly reduced 1,25(OH)2D3 levels (18.1 ± 2.1 and 16.4 ± 2.2 pmol/l compared with 143.5 ± 29 pmol/l for the OVX group), the OVX‐induced decrease in calcium absorption could partially be restored by supplementation with either 0.025 mg or 0.05 mg of 17β‐E2. None of the treatments resulted in a significant change in calcium handling in the jejunum, although the trends were similar as those observed in the duodenum. 17β‐E2 did not change the VDR levels in both the intestine and the kidney. In conclusion, the present study demonstrates that 17β‐E2 is positively involved in intestinal calcium absorption, and the data strengthen the assertion that 17β‐E2 exerts this effect independent of 1,25(OH)2D3. In general, 17β‐E2 not only affects bone turnover but also calcium homeostasis via an effect on intestinal calcium absorption.

Regulation of 1,25-dihydroxyvitamin D3 receptor gene expression by parathyroid hormone and cAMP-agonists

We studied the effect of parathyroid hormone (PTH) and activation of the cAMP signal pathway on vitamin D receptor (VDR) mRNA levels in the phenotypically osteoblast cell line UMR 106. PTH caused a time- and dose-dependent increase of the VDR mRNA content with a maximum after 2 h. After 24 h the VDR mRNA level in PTH-treated cells returned to control level. In contrast, the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-induced increase in VDR mRNA did not decline after 24 h. Inhibition of transcription with actinomycin D (10 micrograms/ml) completely abolished the PTH-induced increase of VDR mRNA and inhibition of translation with cycloheximide (1 microgram/ml) resulted in superinduction of VDR mRNA. The role of cAMP in the induction of VDR mRNA was studied with several agents acting via the cAMP pathway. Incubation for 2 and 4 h with forskolin, Bt2cAMP, PTHrP or prostaglandin E2 caused an increase in the level of VDR mRNA comparable to that caused by PTH. The calcium ionophore A23187 did not affect VDR mRNA level. The present study demonstrates that PTH and activation of the cAMP signal pathway cause up-regulation of VDR via induction of VDR gene expression. The effect of cAMP on the VDR gene is suggestive for a cAMP responsive element in the VDR gene.

Regulation of osteocalcin production and bone resorption by 1,25- dihydroxyvitamin D3 in mouse long bones: Interaction with the bone-derived growth factors TGF-β and IGF-I

Bone cells produce multiple growth factors that have effects on bone metabolism and can be incorporated into the bone matrix. Interplay between these bone‐derived growth factors and calciotropic hormones has been demonstrated in cultured bone cells. The present study was designed to extend these observations by examining the interactions between either transforming growth factor‐β (TGF‐β) or insulin‐like growth factor‐I (IGF‐I) and 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) in a mouse long bone culture model with respect to osteocalcin production and bone resorption. In contrast to the stimulation in rat and human, in the fetal mouse long bone cultures, 1,25(OH)2D3 caused a dose‐dependent inhibition of osteocalcin production. Both the osteocalcin content in the culture medium and in the extracts of the long bones was reduced by 1,25(OH)2D3. This effect was not specific for fetal bone because 1,25(OH)2D3 also reduced osteocalcin production by the neonatal mouse osteoblast cell line MC3T3. TGF‐β inhibited whereas IGF‐I dose‐dependently increased osteocalcin production in mouse long bones. The combination of TGF‐β and 1,25(OH)2D3 did not result in a significantly different effect compared with each of these compounds alone. The IGF‐I effect was completely blocked by 1,25(OH)2D3. In the same long bones as used for the osteocalcin measurements, we performed bone resorption analyses. Opposite to its effect on osteocalcin, 1,25(OH)2D3 dose‐dependently stimulated bone resorption. TGF‐β reduced and IGF‐I did not change basal (i.e., in the absence of hormones) bone resorption. Our results show that 1,25(OH)2D3‐enhanced bone resorption is dose‐dependently inhibited by TGF‐β and IGF‐I. Regression analysis demonstrated a significant negative correlation between 1,25(OH)2D3‐induced bone resorption and osteocalcin production. The specificity for their effect on 1,25(OH)2D3‐stimulated bone resorption was assessed by testing the effects of TGF‐β and IGF‐I in combination with parathyroid hormone (PTH). Like 1,25(OH)2D3, PTH dose‐dependently stimulates bone resorption. However, PTH‐stimulated bone resorption was not affected by TGF‐β. Like 1,25(OH)2D3‐stimulated bone resorption, IGF‐I inhibited the PTH effect but at a 10‐fold higher concentration compared with 1,25(OH)2D3. In conclusion, the present study demonstrates growth factor–specific interactions with 1,25(OH)2D3 in the control of osteocalcin production and bone. With respect to bone resorption, these interactions are also hormone specific. The present data thereby support and extend the previous observations that interactions between 1,25(OH)2D3 and bone‐derived growth factors play an important role in the control of bone metabolism. These data together with the fact that TGF‐β and IGF‐I are present in the bone matrix and potentially can be released during bone resorption support the concept that growth factors may control the effects of calciotropic hormones in bone in a localized and possibly temporal manner. Finally, in contrast to human and rat, in mice 1,25(OH)2D3 reduces osteocalcin production and this reduction is paralleled by stimulation of bone resorption by 1,25(OH)2D3. These data thereby show a dissociation between osteocalcin production and bone resorption.

Consequences of Vitamin D Receptor Regulation for the 1,25-Dihydroxyvitamin D3-Induced 24-Hydroxylase Activity in Osteoblast-like Cells: Initiation of the C24-Oxidation Pathway

A direct relationship between vitamin D receptor (VDR) level and target cell responsiveness to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown in osteoblast-like cell lines. However, we previously found an inverse relationship between the TGF beta-induced VDR up-regulation and subsequent 1,25-(OH)2D3-induced biological responses. A clear inhibition of the 1,25-(OH)2D3-induced stimulation of osteocalcin and osteopontin expression was observed. A biological response that has formerly been shown to be coupled to VDR level is 24-hydroxylase activity. This enzyme initiates the C24 oxidation of the side-chain, followed by cleavage and ultimate metabolic clearance of both 25-(OH)D3 and its metabolite 1,25-(OH)2D3. With UMR 106 (rat) and MG 63 (human) osteoblast-like cells, we show that after preincubation with TGF beta, which causes an increase in VDR level, 1,25-(OH)2D3 induction of 24-hydroxylase activity is also stimulated. In addition, we provide evidence that variations in VDR level induced by other means (PTH, EGF, medium change) are also closely associated with 1,25-(OH)2D3-induced 24-hydroxylase activity. Furthermore, we show that in MG 63 cells, but not in UMR 106 cells, TGF beta itself was able to increase the activity of the enzyme 24-hydroxylase. As 24-hydroxylation is the initial step in the further C24 oxidation of 1,25-(OH)2D3, our results indicate a close coupling of VDR level and the degradation of its ligand, 1,25-(OH)2D3. This mechanism may provide an important regulatory feedback in the action of 1,25-(OH)2D3 at target tissue/cell level.

Transforming growth factor β-induced dissociation between vitamin D receptor level and 1,25-dihydroxyvitamin D3 action in osteoblast-like cells†

Abstract In the present study the interaction between a locally produced factor in bone, transforming growth factor β (TGFβ) and a systemic regulator of bone metabolism, 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ) was investigated. In rat (UMR 106, ROS 17/2.8) and human (MG-63) osteoblastic cell lines and in isolated fetal rat osteoblasts TGFβ caused a comparable increase in vitamin D receptor (VDR) level. A maximum was observed after 6 h at 1 ng/ml TGFβ. Scatchard analysis revealed that up-regulation of VDR is due to an increase in receptor number and not to a change in affinity. This was supported by Northern blot analysis which showed a dose- and time-dependent increase in VDR mRNA by TGFβ. To assess the significance of the TGFβ-induced increase in VDR level for l,25-(OH) 2 D 3 effects cells were preincubated with TGF for 4 h (causing a 2–3-fold increase of the VDR level) and subsequently incubated with l,25-(OH) 2 D 3 for 4 h and 24 h. TGFβ preincubation potently inhibited subsequent l,25-(OH) 2 D 3 stimulation of osteocalcin production in both ROS 17/2.8 and MG-63 cells on protein as well as mRNA level. A similar inhibition by TGFβ was observed on the 1,25-(OH) 2 D 3 -induced increase in osteopontin mRNA. The current study demonstrates dissociation between regulation of VDR level and modulation of two l,25-(OH) 2 D 3 biological responses by TGFβ in osteoblast-like cell lines of different origin. This dissociation shows that, besides interaction at VDR level also at other levels in the cell interaction(s) exist between TGFβ and 1,25-(OH) 2 D 3 . Besides, these data emphasize the potential importance of the interplay of locally produced factors and systemic calciotrophic hormones in the regulation of bone metabolism.

Increasedin vitro release of interleukin-1 by LPS stimulated peritoneal macrophages obtained from patients on continuous ambulatory peritoneal dialysis (CAPD) during an infectious peritonitis

Peritoneal macrophages, collected from dialysate of patients treated with Continuous Ambulatory Peritoneal Dialysis (CAPD) because of end stage renal failure, produce in vitro various mediators such as cyclooxygenase products which in turn stimulates the production of the second messenger cyclic AMP (cAMP), These substances may have inhibitory effects on several functions of mononuclear phagocytes. Earlier we found that when CAPD is complicated by an episode of infectious peritonitis, peritoneal macrophages show a marked decrease in the spontaneous in vitro secretion of cyclooxygenase products as well as in the intracellular cAMP levels in comparison with an infection free period [1]. We postulated that the diminished production of these substances with down regulatory properties would permit the macrophages to release a higher amount of the pro-inflammatory polypeptide interleukin-1 (IL-I) during peritonitis [2]. This mediator plays, together with related cytokines, a key role in the regulation of (acute) inflammatory processes. In the present study we aimed to examine the release of IL-1//, the main secretory form of IL-1, by peritoneal macrophages of CAPD patients, especially with the intention to analyse whether peritoneal macrophages secrete higher amounts of IL-I/~, when CAPD is complicated by an episode of peritonitis. Patients and methods