A combination of MEF3 and NFI proteins activates transcription in a subset of fast-twitch muscles.

The human aldolase A pM promoter is active in fast-twitch muscles. To understand the role of the different transcription factors which bind to this promoter and determine which ones are responsible for its restricted pattern of expression, we analyzed several transgenic lines harboring different combinations of pM regulatory elements. We show that muscle-specific expression can be achieved without any binding sites for the myogenic factors MyoD and MEF2 and that a 64-bp fragment comprising a MEF3 motif and an NFI binding site is sufficient to drive reporter gene expression in some but, interestingly, not all fast-twitch muscles. A result related to this pattern of expression is that some isoforms of NFI proteins accumulate differentially in fast- and slow-twitch muscles and in distinct fast-twitch muscles. We propose that these isoforms of NFI proteins might provide a molecular basis for skeletal muscle diversity.

An opportunistic promoter sharing regulatory sequences with either a muscle-specific or a ubiquitous promoter in the human aldolase A gene.

The human aldolase A gene is transcribed from three different promoters, pN, pM, and pH, all of which are clustered within a small 1.6-kbp DNA domain. pM, which is highly specific to adult skeletal muscle, lies in between pN and pH, which are ubiquitous but particularly active in heart and skeletal muscle. A ubiquitous enhancer, located just upstream of pH start sites, is necessary for the activity of both pH and pN in transient transfection assays. Using transgenic mice, we studied the sequence controlling the muscle-specific promoter pM and the relations between the three promoters and the ubiquitous enhancer. A 4.3-kbp fragment containing the three promoters and the ubiquitous enhancer showed an expression pattern consistent with that known in humans. In addition, while pH was active in both fast and slow skeletal muscles, pM was active only in fast muscle. pM activity was unaltered by the deletion of a 1.8-kbp region containing the ubiquitous enhancer and the pH promoter, whereas pN remained active only in fast skeletal muscle. These findings suggest that in fast skeletal muscle, a tissue-specific enhancer was acting on both pN and pM, whereas in other tissues, the ubiquitous enhancer was necessary for pN activity. Finally, a 2.6-kbp region containing the ubiquitous enhancer and only the pH promoter was sufficient to bring about high-level expression of pH in cardiac and skeletal muscle. Thus, while pH and pM function independently of each other, pN, remarkably, shares regulatory elements with each of them, depending on the tissue. Importantly, expression of the transgenes was independent of the integration site, as originally described for transgenes containing the beta-globin locus control region.

Developmental regulation of the aldolase A muscle-specific promoter during in vivo muscle maturation is controlled by a nuclear receptor binding element.

During the post-natal period, skeletal muscles undergo important modifications leading to the appearance of different types of myofibers which exhibit distinct contractile and metabolic properties. This maturation process results from the activation of the expression of different sets of contractile proteins and metabolic enzymes, which are specific to the different types of myofibers. The muscle-specific promoter of the aldolase A gene (pM) is expressed mainly in fast-twitch glycolytic fibers in adult body muscles. We investigate here how pM is regulated during the post-natal development of different types of skeletal muscles (slow or fast-twitch muscles, head or body muscles). We show that pM is expressed preferentially in prospective fast-twitch muscles soon after birth; pM is up-regulated specifically in body muscles only later in development. This activation pattern is mimicked by a transgene which comprises only the 355 most proximal sequences of pM. Within this region, we identify a DNA element which is required for the up-regulation of the transgene during post-natal development in body muscles. Comparison of nuclear M1-binding proteins from young or adult body muscles show no qualitative differences. Distinct M1-binding proteins are present in both young and adult tongue nuclear extracts, compared to that present in gastrocnemius extracts.

Myotube-specific activity of the human aldolase A M-promoter requires an overlapping binding site for NF1 and MEF2 factors in addition to a binding site (M1) for unknown proteins.

The human aldolase A gene is expressed in several tissues through the use of three alternative promoters. The activity of one of the promoters, pM, is restricted to skeletal muscle. We reported previously that a proximal 280 bp pM fragment confers tissue-specific expression to a CAT reporter gene in transgenic mice. This small regulatory region directs expression to muscle composed mainly of fast-twitch fibers. Here we show that a minimal promoter fragment from base-pairs -164 to +45 is sufficient to highly active pM during myoblast differentiation in cell culture and demonstrate that two DNA elements play a major role in this activation. These elements consist of a binding site (M1) for unknown ubiquitous proteins and an overlapping binding site for MEF2 and NF1 families of transcription factors. The NF1 factor constitute the main binding activity on the MEF2/NF1 site and, interestingly, some of the DNA-protein complexes that form with muscle nuclear extracts on the NF1 element differ from those that form with non-muscular extracts.

Bone resorption by isolated chick osteoclasts in culture is stimulated by murine spleen cell supernatant fluids (osteoclast-activating factor) and inhibited by calcitonin and prostaglandin E2.

The question of whether any of the agents known to activate bone resorption in vivo or in organ cultures acts directly on the osteoclast or via intermediate target cells that secondarily secrete locally paracrine factors is important for our understanding of bone remodeling. In an attempt to clarify this issue for some of the agents, we have taken advantage of the recent progress in obtaining and culturing relatively pure populations of osteoclasts. We performed an in vitro bone-resorbing assay in which isolated and partially purified chick osteoclasts were cultured on devitalized, paired and standardized bone disks prepared from rat calvaria prelabeled with both 45Ca and 3H-proline. Some of the isolated osteoclasts attached to the devitalized bone matrix, formed a ruffled border, and acidified the bone-resorbing compartment that they established with the matrix, thereby indicating that they resorbed bone in a physiologic manner. Salmon calcitonin added to these cultures (0.3 U/ml = 60 ng/ml) and prostaglandin E2 (PGE2) (10(-6) M) inhibited both basal and stimulated 45Ca and 3H-proline release. Neither parathyroid hormone (PTH) 1-34 (1 U/ml), 1,25-(OH)2-D3 (10(-8) and 10(-9) M), nor interleukin 1 (IL-1) (purified from P388D1 macrophage culture supernatant fluids or recombinant murine IL-1-alpha) (100 ng/ml) stimulated bone resorption in these cultures. In contrast, supernatant fluids from concanavalin A (Con-A)-activated murine spleen cell cultures (murine osteoclast-activating factor; OAF) consistently and significantly induced a 3- to 5-fold stimulation of bone resorption in this system.

Uterine cells other than stromal decidual cells are required for 1,25-dihydroxyvitamin D3 production during early human pregnancy.

Human decidual cells are known to produce 1,25-(OH)2D3 at the end of pregnancy, the present study evaluates this capacity, and the part played by stromal decidual cells, in early pregnancy. Cells were obtained from nine human decidua by aspiration or curettage during early pregnancy (7-10 weeks), separated on Ficoll-Paque and plastic adherence, and incubated for 1 h with 25-(OH)D3. Incubation medium and cells were extracted and chromatographed on two successive HPLC systems. The cells examined were of both physiological and pathological (ectopic pregnancy) origin. Endometrial cells obtained in four non-pregnant situations (myomas) were also studied to determine whether the 1,25-(OH)2D3 synthesis by the uterus is associated with the appearance of decidual cells. Results show that human decidual cells from early pregnancy convert 25(OH)D3 (2.5 nM or 2.5 microM) into a metabolite with the physicochemical characteristics of synthetic 1,25-(OH)2D3. This ability is shared by cells isolated during early pregnancy, whether physiological or ectopic (tubal pregnancy). Non-adherent cells, which include mainly stromal decidual cells, are less able to produce 1,25-(OH)2D3 than are the adherent cells, suggesting that macrophages, granulocytes or as yet unidentified cell types are required for the 1,25-(OH)2D3 production by decidual tissue during early human pregnancy. In addition, one out of four experiments with non-pregnant endometrial cells could produce 1,25-(OH)2D3 suggesting that, although not the rule in the non-pregnant state, in vitro production of 1,25-(OH)2D3 by uterine cells can be found in the absence of decidual cells.

Effect of calcitonin in pregnant rats on bone resorption in fetuses.

Fetal bone resorption was measured by an organ culture technique using fetuses from intact or thyroparathyroidectomized pregnant rats. These experiments were performed to investigate the effects of 1,25-dihydroxycholecalciferol (1,25-DHCC) and salmon calcitonin (SCT) in pregnant rats, on both fetal growth and fetal bone resorption. Pregnant rats were given 0.1-0.5 microgram 1,25-DHCC per day from day 17 of gestation: in intact rats bone resorption was increased and fetal growth decreased; 1,25-DHCC probably modified fetal bone resorption in the absence of fetal parathyroid secretion. Infusion of SCT in minipumps (30 mu./h) did not modify plasma calcium levels in either the mother or fetuses, neither was bone resorption altered. In 1,25-DHCC-treated rats, SCT infusion resulted in an increase in fetal weight and a decrease in fetal bone resorption. On the other hand, SCT infusion was found to facilitate phosphate accumulation in fetuses. At the end of the SCT infusion the SCT concentration was 450 ng/l in maternal plasma and 553 +/- 60 ng/l in fetal plasma. Salmon calcitonin was shown to cross the placental barrier in the rats; it may interact with the effects of 1,25-DHCC in the fetus.

Effects of calcitonin on human trophoblastic cells in culture: absence of autocrine control.

The purpose of this work was to investigate the effects of calcitonin (CT) on trophoblastic cells with respect to cAMP levels and human chorionic gonadotrophin (hCG) secretion in cultured cells from first-trimester and term placentas and in a choriocarcinoma cell line (JEG-3). The expression of the CT gene was investigated to elucidate a putative autocrine control of CT during pregnancy. The addition of salmon CT (10(-10) M and above) resulted in concentration-dependent increases in cAMP secretion by normal trophoblastic cells from term and first-trimester placentas. Moreover, CT was found to increase cAMP secretion preferentially in completely differentiated cells, i.e. after 4-7 days in culture. Addition to the culture medium of JEG-3 cells slightly increased cAMP secretion only at a concentration of 10(-8) M. The basal level of hCG in the medium was found to be higher in the first-trimester than in the term trophoblast culture, but salmon CT induced an increase in hCG secretion by term placenta cells only. CT gene expression in our experimental model was investigated to elucidate a putative autocrine control of CT action during pregnancy. It was not found to be expressed in syncytiotrophoblast cells from either first-trimester or term placenta cells by the method used. Our data demonstrate the absence of autocrine control of CT effects in trophoblastic cells, and suggest that CT is likely to exert its effect preferentially on differentiated cells.

Effect of 1 alpha, 24 R,25-trihydroxycholecalciferol on isolated parathyroid cells secretion.

In this work we have studied the action of the trihydroxylated vitamin D metabolite (1 alpha, 24 R,25(OH)3D3) on parathyrin secretion by isolated parathyroid cells. In contrast with the results obtained with mono and dihydroxylated vitamin D, 1 alpha, 24 R,25(OH)3D3 increased the secretion of parathyrin. The effective concentration of this metabolite was 1.54 X 10(-9)M for cells rat and 1.54 X 10(-10)M for human cells.

Comparative effects of some hydroxylated vitamin D metabolites on parathyrin secretion by dispersed rat parathyroid cells in vitro.

We have previously discussed the action of 1 alpha,25-(OH)2D3, (24R) 24,25-(OH)2 D3 and (25S) 25,26-(OH)2D3 on parathyrin secretion by isolated rat parathyroid cells. In this work, we have compared these effects with those obtained with 1 alpha-OH D3, 25-OH D3 and 1 alpha-OH D2. In decreasing order, the activities were: 1 alpha,25-(OH)2D3 greater than 1 alpha-OH D3 greater than (24R) 24,25-(OH)2D3 greater than 25-OH D3 greater than (25S) 25,26(OH)2D3 greater than 1 alpha-OH D2. The presence of two hydroxyl groups with one hydroxyl group in alpha position seems to have the higher activity to inhibit the parathyroid secretion. At least, the nature of the side chain conformation also plays a part upon the effect of PTH release.

Effect of calcitriol on peripheral blood lymphocyte cytotoxicity.

To clarify the biochemical mechanism responsible for the inhibition by calcitriol (1,25-dihydroxyvitamin D3) of cytotoxicity in peripheral blood lymphocytes (PBL), the human NK sensitive K562 cell line and the human tumor necrosis factor-sensitive murine L929 cell line were used as targets and subsequently compared. The cytotoxicity of PBLs for K562 cells was not changed by preincubation for 4 h with 10 ng/ml phorbol myristate acetate (PMA), but was reduced after an overnight preincubation with 10(-9) or 10(-8) M calcitriol. Using L929 cells, preincubation of PBLs with 10 ng/ml PMA for 4 h increased their cytotoxicity. Overnight incubation with calcitriol significantly reduced PBL cytotoxicity for L929 cells in a dose related manner and suppressed the enhancement of this cytotoxicity by PMA. Pretreatment of PBLs with cycloheximide reduced their cytolysis for L929 cells but did not change their cytotoxicity towards K562 cells. Consequently, the natural cytotoxicity of PBLs for K562 cells does not involve the same mechanism as their cytotoxicity for L929 cells and is therefore subject to different forms of regulation. However, calcitriol reduced PBL cytotoxicity towards both target cells.

Effects of 1,25-dihydroxyvitamin D3 on in vitro lymphocyte reactions: arguments for a role at the maternofetal interface.

Calcitriol or 1,25-dihydroxyvitamin D3 is synthesized by successive hydroxylations of vitamin D in the liver and kidney and during pregnancy in the placenta and the decidua. The aim of the present study was to clarify the immunoregulatory role of calcitriol, if any, during pregnancy. Calcitriol concentrations of 10(-10) to 10(-8) M was shown to reduce the proliferation of allogeneically stimulated lymphocytes and cytotoxic cell generation in a dose-dependent manner. However, calcitriol did not inhibit IL-2-dependent proliferation of CTLL-2 cell line. Calcitriol reduced non-MHC restricted cytotoxicity. Calcitriol, therefore, might be involved in the successful engraftment and growth of the fetoplacental unit possibly synergized with other products of placental or decidual origin.

Effect of maternal insulin deficiency on vitamin D metabolite concentrations in normoglycemic pregnant rats and their fetuses.

The effect on vitamin D metabolite concentrations of insulin deficiency, not accompanied by hyperglycemia, were investigated in pregnant rats and in their fetuses injected with 75 mg/kg BW streptozotocin (SZ). These concentrations were measured in maternal plasma and whole fetal body. In the insulinopenic mothers, the 25OHD concentration was found to rise compared with that of control pregnant rats (7.00 +/- 1.66 ng/ml, n = 16, versus control 4.50 +/- 1.60, n = 10, 0.001 less than P less than 0.01). The concentration of 1,25(OH)2D, which was previously found to decrease in pregnant rats that were both hypoinsulinic and hyperglycemic, was previously found to decrease in pregnant rats that were both hypoinsulinic and hyperglycemic, was not different in our control and insulinopenic rats (107.36 +/- 38.25 pg/ml, n = 11, versus control 122.90 +/- 18.20, n = 18.20, n = 8). In fetuses from our SZ-injected rats, the 24,25(OH)2D level diminished compared with the control level (2.12 +/- 0.70 ng/g, n = 11, versus control 5.23 +/- 0.95 ng/g, n = 13, P less than 0.001). The Ca/P ratio in fetal body also decreased (0.68 versus control 1.12). It is suggested that the placental metabolism is an important determinant or normal fetal growth.

Effects of experimental diabetes on the vitamin D metabolism of pregnant rats and their fetuses.

The effects of streptozotocin-induced diabetes on the vitamin D metabolism of pregnant rats were investigated in mothers and their fetuses, 11 and 14 days after streptozotocin (SZ) injection, i.e., on days 18 and 21 of gestation. In the mothers' plasma, the levels of 25-hydroxycholecalciferol (25OHD) and 1,25-dihydroxycholecalciferol (1,25(OH)2 D) were not different from control levels on day 18, but on day 21, 25OHD had increased, 1,25 (OH)2 D had diminished, and significant hypercalcemia was noted (10.1 +/- 0.27 mg/dl vs. 9.47 +/- 0.19 mg/dl, mean +/- SD). In hyperglycemic fetuses from the diabetic mothers, plasma insulin levels were reduced at day 18 but enhanced at day 21. 25OHD levels were not different from those of the controls at day 18, but were lower at day 21 (2.12 +/- 0.70 ng/g BW, n = 13, vs. 3.75 +/- 1.40 ng/g BW n = 29 controls, means +/- SD). Fetal body levels of 1,25 (OH)2 D were lower than that in the controls at day 18 (16.6 +/- 2.9 pg/g BW, n = 9 x 2, vs. 28.7 +/- 6.3 pg/g BW, n = 7 x 2, mean +/- SD P less than 0.001), but identical to control levels on day 21. The role of fetal or placental enzymes in the regulation of vitamin D metabolism in fetuses is discussed.

[Effect of experimental diabetes on levels of 25-hydroxycholecalciferol in pregnant rats and fetuses]. / Effet du diabète expérimental sur les taux de 25-hydroxycholécalciférol chez la rate gestante et le foetus.

Induction of diabetes in female rats by streptozotocin administration 7 days before mating led to an increase in maternal and fetal calcemia at day 21 of gestation. The plasma levels of 25-hydroxycholecalciferol (25 OH D3) were increased in the diabetic mother whereas the 25 OHD3 contents in entire fetuses were greatly decreased in comparison with control values obtained in both normal pregnant rat and normal fetuses. Our results obtained in pregnant rat were different from those found in the literature concerning non pregnant animals in which only 1,25-dihydroxycholecalciferol was affected by diabetes.

Increased metabolic activity of rabbit articular cartilage in vitro.

The metabolic, histochemical and ultrastructural modifications induced in rabbit articular cartilage during in vitro incubation at 37 degrees C, for various periods (10 min to 18 h), using Krebs phosphate-glucose nutrient medium, were studied. It was found that after only 10 min of incubation, the chondrocytes increase their synthesis of matrix macromolecules for, at least, the next 6 h. This was suggested by: 1. Increased incorporation 35S-sulfate and 3H-glycine during the first 6 h of incubation. 2. An intensification of metachromasia, which also spread out into the superficial layer that is normally orthochromatic. Only the most superficial layer corresponding to one or two rows of the cells, retained its staining pattern throughout the incubation; 3. A rapidly acquired abundant rough endoplasmic reticulum, enlarged Golgi area and numerous newly synthesized proteoglycan molecules. This study poses fundamental questions about the mechanisms that regulate matrix synthesis by the chondrocytes.

Phorbol myristate acetate enhances adenylate cyclase activity in human polymorphonuclear leukocytes.

The putative role of protein phosphorylation in modulating adenylate cyclase activity in polymorphonuclear neutrophil membranes was assessed using phorbol myristate acetate (PMA) to stimulate the activity of protein kinase C. PMA was demonstrated to enhance the adenylate cyclase activity stimulated by isoproterenol.

Effect of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 maternal loads on maternal and fetal vitamin D metabolite levels in the rat.

Two groups of female rats were used to investigate vitamin D metabolism in the pregnant animals and in their fetuses. In the first group, 3 micrograms of 25-hydroxyvitamin D3 (25-OH-D3) per kg of body weight were injected into intact or nephrectomized (NX) pregnant rats 3 h before sacrifice on day 21 of pregnancy; in the second group, 2 and 6 ng, respectively, of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) per day were infused continuously into pregnant rats between days 17 and 21 of pregnancy. The findings in the fetuses were obtained by quantitative analysis of extracts (Extrelut) of total fetal body lipids; the extracts were purified on Sep Pak and vitamin D sterols were further separated by high-pressure liquid chromatography. Three hours after the dams were injected with 25-OH-D3, the maternal plasma concentration (mean +/- SD) of 1,25(OH)2D3 was 221 +/- 84 pg/ml. In NX pregnant rats, the 1,25(OH)2D3 levels were still elevated: 95.6 +/- 49.0 pg/ml vs 45 +/- 22 pg/ml in control rats. In fetuses from intact or NX dams, the levels of 25-OH-D3 and 1,25(OH)2D3 were not different from the results obtained in the control fetuses but 24,25(OH)2D3 concentrations were increased (6.7 +/- 1.2 ng vs 2.2 +/- 0.7 ng/g body weight). After maternal infusion of 2 or 6 ng/day of 1,25(OH)2D3 (n = 8), plasma concentrations (mean +/- SD) of the metabolite were 64 +/- 31 and 517 +/- 356 pg/ml, respectively, the second being significantly higher than that of the control rats; 25-OH-D3 and 24,25(OH)2D3 levels did not change. 1,25(OH)2D3 contents (mean +/- SD) in fetuses from the treated dams were not different from those of control fetuses (10 +/- 2 pg/g body weight). Our results suggest that pregnant rats and their fetuses were protected against an excessive increase of 1,25(OH)2D3 concentrations in the maternal plasma; although there was some individual hypercalcemia, no significant increase in mean calcemia was detected in the dams, and 1,25(OH)2D3 either did not cross the placental barrier or was rapidly metabolized because we did not find any changes in the fetal content. As in intact or NX pregnant rats, 25-OH-D3 was metabolized into 1,25(OH)2D3, the increase of 24,25(OH)2D3 in the fetuses might be associated with a protective mechanism.

Metabolism of human femoral head cartilage in osteoarthrosis and subcapital fracture.

The cell density and incorporation of 35SO4 and 3H-glycine into human articular cartilage from 8 osteoarthrotic and 7 normal (subcapital fracture) femoral heads were studied. It was found that osteoarthrotic cartilage incorporates on a per cell basis about twice as much 35SO4 and 2--5 times as much 3H-glycine as normal cartilage. There was no relationship between the intensity of incorporation and either the location of the cartilage (weight-bearing versus non weight-bearing areas) in normal cartilage or the degree of damage (normal-like, fibrillated, and ulcerated) in osteoarthrotic articular cartilage. In the latter tissue the increased synthetic capacity of the cells seems to be a diffuse rather than a localised process, for it was also found in cartilage from peripheral osteophytes. Histo-autoradiographic studies showed that the osteoarthrotic chondrocytes are metabolically hyperactive all over the femoral head, including wedge-shaped margins of the zone of exposed bone. These results support the hypothesis that much of the articular cartilage from osteoarthrotic femoral heads is of an immature chondroblastic type. It is suggested that de-novo synthesis of articular cartilage occurs during the process of regional remodelling of the femoral head, which would account for the observed hyperactivity.

[Effects of the dihydroxyl metabolites of vitamin D3 and their ethanol solvent on parathyroid secretion: in vivo study in the rat]. / Effets des métabolites dihydroxyles de la vitamine D3 et de leur solvant éthanol sur la sécrétion parathyroïdienne: étude in vivo chez le rat.

The regulation of parathyroid secretion by the vitamin D3 dihydroxylated metabolites was studied by differents authors. In vitro experiments showed that 1 alpha (OH)2 D3 and 24 R 25 (OH)2 D3 inhibited the PTH release. In Rats maintained in a normal calcium diet or calcium and/or vitamin D deficient diet, 1 alpha 25(OH)2 D3 and 24 R 25 (OH)2 D3 inhibited the PTH release, whereas 24 S 25 (OH)2 D3, 25 S 26 and 25 R 26(OH)2 D3 had no effect.