The expression of osteoprotegerin and RANK ligand and the support of osteoclast formation by stromal-osteoblast lineage cells is developmentally regulated

The one or more molecular mechanisms that determine the obligatory sequence of resorption followed by formation during bone remodeling is unclear. RANK ligand (RANK-L) is an essential requirement for osteoclastogenesis, and its activity is neutralized by binding to the soluble decoy receptor, osteoprotegerin (OPG). Because both molecules are produced by osteoblast lineage cells, we studied their developmental regulation in a conditionally immortalized human marrow stromal (hMS[2-15]) cell line. These cells can simulate the complete developmental sequence from undifferentiated precursor(s) to cells with the complete osteoblast phenotype that are capable of forming mineralized nodules. During osteoblast differentiation, RANK-L messenger RNA levels decreased by 5-fold, whereas OPG messenger RNA levels increased by 7-fold, resulting in a 35-fold change in the RANK-L/OPG ratio. OPG protein also increased by 6-fold. Mouse bone marrow cells generated osteoclast-like cells in coculture with undifferentiated hMS(2-15) cells, but did not when cocultured with hMS(2-15) cells in varying stages of differentiation, unless an excess of RANK-L was added. Thus, undifferentiated marrow stromal cells with a high RANK-L/OPG ratio can initiate and support osteoclastogenesis, but after differentiation to the mature osteoblast phenotype, they cannot. We speculate that the developmental regulation of OPG and RANK-L production by stromal/osteoblast cells contributes to the coordinated sequence of osteoclast and osteoblast differentiation during the bone remodeling cycle.     

Estrogen promotes early osteoblast differentiation and inhibits adipocyte differentiation in mouse bone marrow stromal cell lines that express estrogen receptor (ER) alpha or beta

Although cells of the osteoblast lineage express functional ERs, direct effects of estrogen on bone formation remain obscure. In the present study, we have investigated estrogen effects on osteoblastic and adipocytic differentiation from a mouse bone marrow stromal cell line, ST-2, which had been manipulated to overexpress either human ER alpha (ST2ER alpha) or ER beta (ST2ER beta). Treatment with bone morphogenetic protein-2 increased alkaline phosphatase activity as well as the number of Oil Red O-positive adipocytes, indicating that bone morphogenetic protein-2 stimulated both osteoblastic and adipocytic differentiation from these bipotential cells. In both ST2ER alpha and ST2ER beta cells, cotreatment with E2 caused enhancement of alkaline phosphatase activity and suppression of lipid accumulation. These effects were completely reversed by an ER antagonist, ICI182780. Therefore, the estrogen regulation occurred in an ER-specific manner but without ER subtype specificity. Moreover, dose response curves of the opposing effects of estrogen on osteoblastogenesis and adipogenesis formed an apparent mirror image, consistent with a reciprocal regulation of differentiation into the two cell lineages. These results demonstrate that estrogen directly modulates differentiation of bipotential stromal cells into the osteoblast and adipocyte lineages, causing a lineage shift toward the osteoblast. Such effects would lead to direct stimulation of bone formation and thereby contribute to the protective effects of estrogen on bone.     

淫羊藿总黄酮对去卵巢大鼠骨髓间充质干细胞成骨和成脂分化中DKK1蛋白动态表达的影响

目的观察DKK1在淫羊藿总黄酮调控去势雌性大鼠骨髓间充质干细胞成骨和成脂分化平衡过程中的动态表达,为进一步阐明淫羊藿总黄酮治疗绝经后骨质疏松症的作用机制提供实验依据。方法体外分离培养去势雌性大鼠来源骨髓间充质干细胞,分别在成骨诱导液和脂肪诱导液条件下连续培养15 d,并在此基础上添加剂量为10μg/mL的淫羊藿总黄酮。采用ALP染色、ALP活性测定、油红O染色以及荧光定量PCR技术,观察淫羊藿总黄酮对骨髓间充质干细胞成骨和成脂分化的影响。用酶联免疫法(ELISA)检测淫羊藿总黄酮处理过程中DKK1蛋白的动态表达,观察DKK1蛋白在淫羊藿总黄酮调控去势雌性大鼠骨髓间充质干细胞成骨和成脂分化平衡过程中的作用。结果淫羊藿总黄酮能显著增加骨髓间充质干细胞ALP的表达以及成骨早期分化因子Runx2 mRNA的表达,显著下调骨髓间充质干细胞中脂肪形成关键基因PPARγ-2mRNA的表达,从而抑制脂滴的形成。在成骨诱导条件下,EFs呈时间依赖性下调DKK1的表达;在脂肪诱导条件下,EFs呈时间依赖性抑制DKK1蛋白的上调。结论通过抑制DKK1蛋白的表达调控去势雌性大鼠BMSCs成骨和成脂分化平衡,可能是淫羊藿总黄酮治疗绝经后骨质疏松症的分子机制之一。     

IL-3 is a potential inhibitor of osteoblast differentiation in multiple myeloma

Bone destruction in multiple myeloma is characterized both by markedly increased osteoclastic bone destruction and severely impaired osteoblast activity. We reported that interleukin-3 (IL-3) levels are increased in bone marrow plasma of myeloma patients compared with healthy controls and that IL-3 stimulates osteoclast formation. However, the effects of IL-3 on osteoblasts are unknown. Therefore, to determine if IL-3 inhibits osteoblast growth and differentiation, we treated primary mouse and human marrow stromal cells with IL-3 and assessed osteoblast differentiation. IL-3 inhibited basal and bone morphogenic protein-2 (BMP-2)-stimulated osteoblast formation in a dose-dependent manner without affecting cell growth. Importantly, marrow plasma from patients with high IL-3 levels inhibited osteoblast differentiation, which could be blocked by anti-IL-3. However, IL-3 did not inhibit osteoblast differentiation of osteoblastlike cell lines. In contrast, IL-3 increased the number of CD45+ hematopoietic cells in stromal-cell cultures. Depletion of the CD45+ cells abolished the inhibitory effects of IL-3 on osteoblasts, and reconstitution of the cultures with CD45+ cells restored the capacity of IL-3 to inhibit osteoblast differentiation. These data suggest that IL-3 plays a dual role in the bone destructive process in myeloma by both stimulating osteoclasts and indirectly inhibiting osteoblast formation.     

Leptin reduces ovariectomy-induced bone loss in rats.

Bone mineral density increases with fat body mass, and obesity has a protective effect against osteoporosis. However, the relationship between fat body mass and bone mineral density is only partially explained by a combination of hormonal and mechanical factors. Serum leptin levels are strongly and directly related to fat body mass. We report here the effects of leptin administration compared with estrogen therapy on ovariectomy-induced bone loss in rats. Leptin was effective at reducing trabecular bone loss, trabecular architectural changes, and periosteal bone formation. Interestingly, the combination of estrogen and leptin further decreased bone turnover compared with that in estrogen-treated ovariectomized rats. Leptin also significantly increased osteoprotegerin mRNA steady state levels and protein secretion and decreased RANK ligand mRNA levels in human marrow stromal cells in vitro. Our findings suggest that leptin could modulate bone remodeling in favor of a better bone balance in rats. This study is the first evidence that leptin therapy has a significant effect in preventing ovariectomy-induced bone loss, and this effect may at least in part be mediated by the osteoprotegerin/RANK ligand pathway.     

Divergent effects of selective peroxisome proliferator-activated receptor-gamma 2 ligands on adipocyte versus osteoblast differentiation

PPAR gamma is activated by diverse ligands and regulates the differentiation of many cell types. Based on evidence that activation of PPAR gamma 2 by rosiglitazone stimulates adipogenesis and inhibits osteoblastogenesis in U-33/gamma 2 cells, a model mesenchymal progenitor of adipocytes and osteoblasts, we postulated that the increase in marrow fat and the decrease in osteoblast number that occur during aging are due to increased PPAR gamma 2 activation. Here, we show that the naturally occurring PPAR gamma ligands 9,10-dihydroxyoctadecenoic acid, and 15-deoxy-Delta(12,14)-PGJ(2), also stimulate adipocytes and inhibit osteoblast differentiation of U-33/gamma 2 cells. Strikingly, 9,10-epoxyoctadecenoic acid and the thiazolidine acetamide ligand GW0072 [(+/-)-(2S,5S)-4-(4-(4-carboxyphenyl)butyl)-2-heptyl-4-oxo-5-thaizolidineN,N-dibenzyl-acetamide] prevent osteoblast differentiation, but do not stimulate adipogenesis, whereas 9-hydroxyoctadecadienoic acid stimulates adipogenesis but does not affect osteoblast differentiation. The divergent effects of PPAR gamma 2 ligands on osteoblast and adipocyte differentiation were confirmed in primary murine bone marrow cultures using rosiglitazone and GW0072. These findings indicate that the proadipogenic and antiosteoblastogenic effects of PPAR gamma 2 are mediated by distinct regulatory pathways that can be differentially modulated depending on the nature of the ligand, and they support the idea that increased fatty acid oxidation during aging may inhibit osteoblast differentiation. Moreover, there may be selective PPAR gamma 2 modulators that block the adverse effects of fatty acid oxidation products while retaining beneficial activities such as insulin sensitization.     

Synergistic actions of insulin-sensitive and Sirt1-mediated pathways in the differentiation of mouse embryonic stem cells to osteoblast

Murine embryonic stem cells (mESCs) have the potential to differentiate into almost any type of cell, and hence, represent a useful biological resource for tissue engineering. The differentiation of mESCs into osteoblasts in vitro is usually dampened by simultaneous differentiation of adipocytes. Insulin exerts a profound effect on bone development through increased differentiation of osteoblasts and concurrent formation of adipocytes. Comparatively, Sirt1, which plays a crucial role in osteoblast differentiation, has been reported to down regulate adipocyte formation during osteoblast differentiation. This study analyzed the combined effects of insulin and Sirt1 on the differentiation of osteoblasts. Osteoblast differentiation was quantified by estimating the accumulation of mineralized matrix and expression of osteogenic genes. The present data show that the simultaneous action of the insulin and Sirt1-mediated pathways increased the efficiency of osteoblast differentiation. When the cells were tested for ALP activity and Alizarin red staining, there was a respective increase of ~180% and ~166% (P<0.05) compared to the control. Furthermore, the mRNA expression patterns of osteoprotegerin, osterix, runx2, and osteopontin were increased by 3.6, 2.3, 1.8, and 1.7-fold, respectively, with a concomitant decrease in the mRNA expression levels of adipocyte marker genes. Interestingly, blocking the effects of both Sirt1 and insulin resulted in decreased osteoblastogenesis (60%) and subsequent increased adipocyte differentiation (195%) (P<0.05). Moreover, immunoblotting analysis demonstrated that this activation was via an Akt-dependent pathway. In conclusion, the present data suggests an enhanced process of osteoblast differentiation that can be exploited further to improve mESC differentiation.     

High concentration of leptin stimulates myeloid differentiation from human bone marrow CD34+ progenitors: potential involvement in leukocytosis of obese subjects

OBJECTIVE AND DESIGN: As well as its involvement in control of adipose mass and body energy balance, several reports suggest a link between leptin and hemopoiesis. To test its putative role in human hemopoiesis, we developed a homologous system, ie recombinant human leptin treatment of purified CD34+ progenitors from adult human bone marrow. RESULTS: Leptin (50-100 ng/ml) significantly stimulated the appearance of granulocyte-macrophage colonies in the presence or absence of erythropoietin. The concentration of leptin required for this effect was rather high but within the range of plasma leptin levels observed in obesity. Two results further support the hypothesis that leptin may be involved in the leukocytosis associated with obesity: (i) leptin concentrations in bone marrow and plasma of subjects studied were highly correlated; (ii) leptin and leukocyte count were correlated only in obese subjects. Paracrine effects of locally released leptin from bone marrow adipocytes could also be involved in the regulation of hemopoiesis, a hypothesis supported by marrow immunocytochemistry revealing the close association of CD34+ cells with adipocytes and by previous demonstration that leptin is secreted at a high level by these cells. CONCLUSION: These results indicate that leptin acts on human multilineage CD34+ cells and that high plasma leptin levels associated with obesity could participate in the differentiation of granulocytes from hemopoietic progenitors.     

Aromatase activity of human mesenchymal stem cells is stimulated by early differentiation, vitamin D and leptin

Human mesenchymal stem cells (hMSCs) are multipotent cells present in bone marrow, which differentiate into osteoblasts and adipocytes, among other lineages. Oestrogens play a critical role in bone metabolism; its action may affect the adipocyte to osteoblast ratio in the bone marrow. In hMSCs, oestrogens are synthesized from C19 steroids by the enzyme aromatase cytochrome P450. In this study, we assessed whether aromatase enzymatic activity varied through early osteogenic (OS) and adipogenic (AD) differentiation. Also, we studied the effect of leptin and 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) on aromatase cell activity. Finally, we analysed whether conditions that modify oestrogen generation by cells affected hMSCs differentiation. For these purposes, hMSCs derived from post-menopausal women (65-86 years old) were cultured under basal, OS or AD conditions, in the presence or the absence of leptin and 1,25(OH)2D3. Aromatase activity was measured by the tritiated water release assay and by direct measurement of steroids synthesized from 3H-labelled androstenedione or testosterone. Our results showed that different OS and AD patterns of aromatase activity developed during the first period of differentiation (up to 7 days). A massive and sharp surge of aromatase activity at 24 h characterized early OS differentiation, while increased but constant aromatase activity was increased through adipogenesis. Both leptin and vitamin D increased aromatase activity during osteogenesis, but not during adipogenesis; finally, we showed that favourable aromatase substrates concentration restrained MSCs adipogenesis but improved osteogenesis. Thus, it could be inferred that a high and early increase of local oestrogen concentration in hMSCs affects their commitment either restraining AD or facilitating OS differentiation, or both.     

The phytoestrogen genistein enhances osteogenesis and represses adipogenic differentiation of human primary bone marrow stromal cells

In the present study, we investigated the role of the phytoestrogen genistein and 17beta-estradiol in human bone marrow stromal cells, undergoing induced osteogenic or adipogenic differentiation. Profiling of estrogen receptors (ERs)-alpha, -beta1, -beta2, -beta3, -beta4, -beta5, and aromatase mRNAs revealed lineage-dependent expression patterns. During osteogenic differentiation, the osteoblast-determining core binding factor-alpha1 showed a progressive increase, whereas the adipogenic regulator peroxisome proliferator-activated receptor gamma (PPARgamma) was sequentially decreased. This temporal regulation of lineage-determining marker genes was strongly enhanced by genistein during the early osteogenic phase. Moreover, genistein increased alkaline phosphatase mRNA levels and activity, the osteoprotegerin:receptor activator of nuclear factor-kappaB ligand gene expression ratio, and the expression of TGFbeta1. During adipogenic differentiation, down-regulation in the mRNA levels of PPARgamma and CCAAT/enhancer-binding protein-alpha at d 3 and decreased lipoprotein lipase and adipsin mRNA levels at d 21 were observed after genistein treatment. This led to a lower number of adipocytes and a reduction in the size of their lipid droplets. At d 3 of adipogenesis, TGFbeta1 was strongly up-regulated by genistein in an ER-dependent manner. Blocking the TGFbeta1 pathway abolished the effects of genistein on PPARgamma protein levels and led to a reduction in the proliferation rate of precursor cells. Overall, genistein enhanced the commitment and differentiation of bone marrow stromal cells to the osteoblast lineage but did not influence the late osteogenic maturation markers. Adipogenic differentiation and maturation, on the other hand, were reduced by genistein (and 17beta-estradiol) via an ER-dependent mechanism involving autocrine or paracrine TGFbeta1 signaling.     

Recombinant human bone morphogenetic protein-2 induces osteoblastic differentiation in W-20-17 stromal cells.

To better understand the in vivo bone-inductive properties of recombinant human (rh) BMP-2, we examined the ability of the protein to alter the phenotype of a bone marrow stromal cell line. W-20-17. rhBMP-2 increased alkaline phosphatase activity in W-20-17 cells in a dose-responsive manner in the absence of an effect on proliferation. The induction of alkaline phosphatase activity was not apparent until 12 h after rhBMP-2 treatment had begun and was effectively eliminated by cotreatment with cycloheximide, suggesting a requirement for protein synthesis. Continued treatment of W-20-17 cells with rhBMP-2 for 8 days resulted in a significant increase, compared to control cultures, in the production of cellular cAMP in response to a PTH challenge. In addition, 4-day treatment with rhBMP-2 induced osteocalcin levels in W-20-17 cells. These results indicate that rhBMP-2 induces the expression of several markers associated with the osteoblast phenotype in W-20-17 cells and raises the possibility that BMP-2 may be involved in the differentiation of osteoblasts from progenitor cells resident in bone marrow.     

Effects of BMP-2 on osteoblastic cells and on skeletal growth and bone formation in unloaded rats.

A previous study showed that skeletal unloading induced by hindlimb suspension for 14 days in rats reduces osteoblastic cell proliferation, inhibits skeletal growth and bone formation and induces metaphyseal bone loss. This study investigated the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) in this model. In vitro analysis showed that rhBMP-2 (25-100 ng/ml, 48-96 h) increased alkaline phosphatase activity, an early marker of osteoblast differentiation, in rat neonatal calvaria cells and adult marrow stromal cells, showing that rhBMP-2 induced the differentiation of osteoblast precursor cells in vitro. In contrast, rhBMP-2 did not increase rat calvaria or marrow stromal cell proliferation. Biochemical and histomorphometric analysis showed that systemic infusion with rhBMP-2 (2 microg/kg/day) in unloaded rats had no significant effect on serum osteocalcin levels and on histomorphometric indices of bone formation. Accordingly, rhBMP-2 infusion did not prevent the decreased skeletal growth, trabecular bone bone volume and bone mineral content induced by unloading. The present data indicate that, although rhBMP-2 stimulates osteoblastic cell differentiation, rhBMP-2 infusion is not effective in increasing bone formation and in preventing trabecular bone loss induced by unloading in rats.     

Differentiation of human mesenchymal stem cells: the potential mechanism for estrogen-induced preferential osteoblast versus adipocyte differentiation

IntroductionBone marrow-derived mesenchymal stem cells (MSCs) are capable of differentiating into osteoblasts and adipocytes. This critical balance between osteoblast and adipocyte differentiation plays a significant role in maintaining normal bone homeostasis. In osteoporosis, a metabolic bone disease seen mainly in postmenopausal women because of estrogen deficiency, the concomitant occurrence of increased bone marrow adipocyte production with diminished production of osteoblasts, points to the potential role of estrogen in shifting the balance of MSC differentiation.MethodsWe established an in vitro differentiation model of isolated human MSCs (hMSCs) and examined the role of distinct estrogen signaling pathways in regulating the differentiation of hMSCs.ResultsEstrogen promoted the differentiation of hMSCs to osteoblasts in contrast to adipocytes, the former of which was mediated through the PI3K/SSH1L but not the mitogen-activated protein kinase pathway.ConclusionThis study provides a novel mechanistic understanding of estrogen-related osteoporosis and identifies potential targets for antiosteoporosis therapies.     

Retinoids (all-trans and 9-cis retinoic acid) stimulate production of macrophage colony-stimulating factor and granulocyte-macrophage colony- stimulating factor by human bone marrow stromal cells

Retinoic acids (RAs) exert pleiotropic effects on cellular growth and differentiation. All-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA), a stereoisomer of ATRA, induce differentiation of leukemic cell lines and cells from patients with acute myelogenous leukemia (AML) in vitro. Despite information on the effects of RAs on hematopoietic cells, little is known about how RAs act on the hematopoietic microenvironment, especially on bone marrow stromal cells. Based on recent observations that various cytokines produced mainly by bone marrow stromal cells regulate hematopoiesis, we analyzed the effects of RAs on cytokine production by these cells. ATRA or 9-cis RA treatment of human bone marrow stromal cell line KM101, which produces macrophage colony-stimulating factor (M-CSF) and granulocyte- macrophage colony-stimulating factor (GM-CSF) constitutively, enhanced mRNA levels of both cytokines in a dose-dependent manner. Both RAs also stimulated M-CSF production from primary cultures of human bone marrow stromal cells. Both retinoic acid receptor (RAR)-alpha and retinoid X receptor (RXR)-alpha were expressed constitutively in KM101 cells. ATRA did not affect the expression of either receptor, whereas 9-cis RA increased RXR-alpha mRNA expression in a dose-dependent manner, but did not affect levels of RAR-alpha mRNA. These findings may have important biologic implications for both the role of RAs in hematopoiesis and the therapeutic effects of ATRA on the hematopoietic microenvironment in patients with acute promyelocytic leukemia (APL).     

Thiazolidinediones inhibit osteoclast-like cell formation and bone resorption in vitro.

Osteoblasts and adipocytes are derived from common bone marrow stromal cells that play crucial roles in the generation of osteoclasts. Activation of peroxisome proliferator-activated receptor-gamma (PPARgamma) induces adipogenic differentiation of stromal cells; however, whether this would affect osteoblast/osteoclast differentiation is unknown. Thus, we examined the effects of the thiazolidinedione (TZD) class of antidiabetic agents that activate PPARgamma on osteoblast/osteoclast differentiation using mouse whole bone marrow cell culture. As reported, all TZDs we tested (troglitazone, pioglitazone, and BRL 49653) markedly increased the number of Oil Red O-positive adipocytes and the expression of adipsin and PPARgamma 2. 1alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] did not affect adipogenic differentiation induced by TZDs. TZDs did not affect alkaline phosphatase activity, an early marker of osteoblastic differentiation, despite their marked adipogenic effects. TZDs decreased the number of tartrate-resistant acid phosphatase-positive multinucleated osteoclast-like cells induced by 1,25-(OH)2D3 or PTH. Troglitazone dose dependently inhibited basal and 1,25-(OH)2D3- and PTH-induced bone resorption as assessed by pit formation assay. Interleukin-11 blocked the induction by troglitazone of adipogenesis, but had no effect on the inhibition of osteoclast-like cell formation. These results indicate that TZDs are potent inhibitors of bone resorption in vitro. Inhibitory effects of TZDs on osteoclastic bone resorption was not osteotropic factor specific and did not appear to be related to their adipogenic effects. Thus, TZDs may suppress bone resorption in diabetic patients and prevent bone loss.     

Bone marrow and peripheral blood leptin levels in lymphoproliferative diseases--relation to the bone marrow fat and infiltration

Leptin is a nonglycosylated protein produced mostly by adipocytes. The role ofleptin in body weight regulation through its anorectic effect in hypothalamus is very well known. Less known are other leptin effects such as the stimulation of hematopoesis and some parts of immunity system. The role of leptin in the pathogenesis of some malignant tumors is discussed. Only a little is known about bone marrow adipocyte leptin production. We examined leptin concentrations in the sera from peripheral blood and bone marrow, the percentage of bone marrow fat, the degree of bone marrow infiltration, the body mass index (BMI) in 42 patients with lymphoproliferative diseases. We found that bone marrow has significantly lower leptin levels (6,6+/-10,9 ng/ml) than peripheral blood (9,1+/-11,5 ng/ml) (p < 0.0001). Bone marrow and peripheral blood leptin levels have also a significant thin correlation (r = +0.91, p < 0.0001). Bone marrow (r = +0.55, p < 0.0005) and peripheral blood (r = +0.52, p < 0.0005) leptin concentrations are significantly correlated to BMI. Blood serum leptin (r = +0.46, p < 0.003) and bone marrow leptin (r = +0.40, p < 0.01) are related to the bone marrow fat percentage. In addition we found a negative correlation of blood serum leptin (r = -0.59, p < 0.0001) and bone marrow leptin (r = -0.42, p < 0.005) to bone marrow malignant infiltration. When we divided the patients into groups with bone marrow infiltration more than 10% and without or less than 10% infiltration, the first group had significantly lower peripheral blood (p < 0.001) and bone marrow (p < 0.02) leptin. We also confirmed a relation of bone marrow fat and infiltration (r = +0.49, p < 0.001). Our results suggest a relationship among leptin levels in blood or bone marrow and bone marrow infiltration in lymphoproliferative diseases. This fact needs further investigation and an evaluation of its application in clinical practice.     

Leptin and Ob-Rb receptor isoform in the human digestive tract during fetal development

CONTEXT: Leptin, partially produced by the stomach, is a hormone involved in energy balance and regulation of food intake. It also regulates some digestive functions through its functional receptor Ob-Rb expressed by gastrointestinal epithelial cells. OBJECTIVE: The objective of the study was to investigate the temporal and spatial appearance of Ob-Rb in the human digestive tract and leptin in the stomach. DESIGN: The esophagus, stomach, and intestine samples of 7- to 24-wk-old human fetuses and adult mucosae were studied by RT-PCR, immunohistochemistry, and Western blot. Leptin was measured by RIA in amniotic fluids at 16-33 wk gestation. RESULTS: All mucosae expressed Ob-Rb (mRNA and/or protein) between 7 and 9 wk gestation. Leptin protein appeared by 8 wk in the gastric mucosa, whereas leptin mRNA was detected around 11 wk. Leptin levels in amniotic fluids were significantly higher during the second than during the third trimester. Overall, Ob-Rb immunoreactivity was higher in young fetuses, during the period corresponding to the formation of gastric buds and primitive intestinal crypts and the beginning of differentiation of epithelial cell types, than in the oldest. Leptin added to culture medium of gastrointestinal explants from 10- to 12-wk-old fetuses appeared to affect DNA synthesis as compared with controls, indicating that leptin receptor functionality was developing. CONCLUSIONS: The strong expression of leptin, in amniotic fluid when fetuses begin swallowing then in the gastric mucosa, and the early presence of Ob-Rb in mucosae suggest a possible role for leptin, exerted endoluminally and in a paracrine pathway, in the developmental process (growth and/or maturation) of the human digestive tract.