Osteogenic potential of rat spleen stromal cells

Evidence is mounting that an increasing number of cell populations in the adult organism already committed and/or differentiated retain the ability to reprogram themselves and give rise to a different phenotype. Bone marrow stromal cells have long been recognized as early progenitor cells for osteoblasts, chondrocytes, hematopoietic-supportive fibroblasts and adipocytes. Recent reports though have demonstrated a potential of cell populations outside the bone marrow environment to sustain bone formation under specific circumstances. The formation of bone nodules in the spleen of IL-5 transgenic mice has been recently reported (Macias et al. (2001): J. Clin. Invest. 107, 949 - 959). We thus postulated that a cell population exists in the spleen that under particular microenvironmental conditions is able to reprogram itself and pursue a fate other than the tissue-specific one. Therefore we isolated and expanded in vitro spleen-derived stromal cells. After expansion, these cells were challenged with culture conditions designed to induce osteogenic differentiation. We hypothesized that the combination of a proliferating factor (fibroblast growth factor 2) and a differentiating hormone (dexamethasone) would allow us to induce spleen-derived stromal cells to proliferate and at the same time to express osteoblast-specific genes. Thus, spleen-derived stromal cells were isolated from rat spleen and expanded in the presence of fibroblast growth factor 2 and dexamethasone. Once primary cultures reached confluence they were either switched to an osteo-inductive medium or implanted in immunodeficient mice. Although no bone formation was observed in in vivo experiments, in vitro spleen-derived stromal cells were able to deposit a mineralized matrix. Gene expression, as revealed by RT-PCR analysis, evidenced that the deposition of a mineralized matrix was concomitant with the expression of CBFA1 and osteocalcin, along with alkaline phosphatase and bone sialoprotein. Our data suggest that rat spleen-derived stromal cells can undergo osteogenic differentiation in a permissive microenvironment.     

Possible involvement of protein kinase C in the induction of adipose differentiation-related protein by Sterol ester in RAW 264.7 macrophages

We studied the effects of BMP-7/OP-1 on growth and differentiation of bone marrow stromal cells. BMS2, a mouse bone marrow stromal cell line capable of differentiating into adipocytes and osteoblasts, were treated in a serum-free medium containing differentiation agents that favor the expression of both lineages. BMP-7/OP-1 stimulated cell proliferation and differentiation concomitantly. These effects were dose- and growth phase-dependent. Cells were more sensitive to the treatment early in the culture (30-40% confluence) with a significant increase in cell proliferation and markers of differentiation at low concentrations. When treated later in the growth phase (90-100% confluence), no significant increase in cell proliferation was seen. The concentration requirement for cells later in the culture to reach an equivalent degree of differentiation was 3-10- fold higher than for cells treated early. In both cases, the effects on adipocyte differentiation were biphasic; low concentrations stimulated adipocyte differentiation which was inhibited at higher concentrations where stimulation of osteoblast markers were observed. We conclude that cell proliferation and cell differentiation into adipocyte/osteoblast can occur simultaneously under BMP-7/OP-1 treatment.     

Effects of Growth Hormone on the Function of β‐Adrenoceptor Subtypes in Rat Adipocytes

Objective: The influence of growth hormone (GH) on the regulation of lipolytic response to specific agonists to β‐adrenoceptors and several post‐receptor steps in the lipolytic cascade were investigated. Research Methods and Procedures: Adipose tissues from rats were incubated with or without GH (1.38 nM). After a 24‐hour incubation, isolated adipocytes were prepared for different assays. Rats were hypophysectomized. One week after operation, l‐thyroxine and hydrocortisone acetate was given to hypophysectomized rats. One group of rats was treated with GH (1.33 mg/kg, daily). After 1 week of hormonal treatment, adipose tissues were removed for different studies. Results: GH treatment increased both basal lipolysis and lipolytic sensitivity to dobutamine and CGP 12177 in adipocytes. The lipolytic sensitivity to terbutaline was not influenced by GH treatment. GH treatment increased the maximal lipolytic response to dobutamine and CGP 12177, but not to terbutaline as determined with absolute values of lipolysis. Forskolin‐induced lipolysis was increased by addition of GH to tissues. Moreover, GH treatment resulted in enhanced expression of hormone‐sensitive lipase. GH treatment in hypophysectomized rats influenced neither the expressions of Gαs protein and cholera toxin‐catalyzed adenosine diphosphate‐ribosylation of Gαs protein, nor cholera toxin‐induced 3′, 5′‐cyclic adenosine monophosphate accumulation. However, the expression of Gαi protein was decreased after GH treatment. Discussion: These and previous results suggest that GH increases lipolysis in rat adipocytes partly through the β‐adrenergic system, including increases in both β₁‐ and β₃‐adrenergic receptor function, and partly through enhanced adenylate cyclase function, and expression of hormone‐sensitive lipase, perhaps via a decrease in Gαi protein expression.     

Insulin-like growth factor I and erythropoiesis.

The bone marrow, the primary site of hematopoiesis, is a self-renewing system consisting of a unique micro-environment that promotes the differentiation and proliferation of the various hematopoietic cell lines. While many critical factors necessary for red cell production have been identified, the regulation of erythropoiesis has not been completely elucidated. In addition to multi-lineage growth factors (e.g. interleukin 3 or 4) and lineage-specific hematopoietic growth factors (e.g. erythropoietin), several lines of evidence suggest a key role for insulin-like growth factor I (IGF-I). First, growth hormone stimulates erythropoiesis and IGF-I is known to mediate many of growth hormone's actions (somatomedin hypothesis). Second, factors in bovine serum and in serum from an anephric human with erythropoietic activity distinct from erythropoietin have been identified as IGFs. Third, IGF receptors are found on both erythrocyte precursors as well as mature erythrocytes. Fourth, in vitro IGF-I stimulates erythropoiesis in bone marrow cultures. Fifth, IGF-I administration to neonatal or hypophysectomized animals results in increased erythropoiesis in vivo. Recent studies indicate that IGF-I at physiologic concentrations stimulates erythropoiesis and that growth hormone's action is indirect, occurring via IGF-I. The physiologic source of IGF-I for the bone marrow may be delivery from the serum (an endocrine mechanism) or synthesis within the bone marrow by stromal or other cells (a paracrine mechanism). Our recent studies have shown that mouse bone marrow stromal cells secrete both IGF-I and IGF binding proteins (IGFBPs). The role of IGFBPs in erythropoiesis is not known, but they might modulate the local concentration of IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Injections of leptin into rat ventromedial hypothalamus increase adipocyte apoptosis in peripheral fat and in bone marrow

The accumulation of fat cells (adipocytes) in bone marrow is now thought to be a factor contributing to age-related bone loss. Women with osteoporosis have higher numbers of marrow adipocytes than women with healthy bone, and bone formation rate is inversely correlated with adipocyte number in bone tissue biopsies from both men and women. Adipogenic differentiation of bone marrow stromal cells increases with age, but the factors regulating populations of mature adipocytes are not well understood. Leptin is thought to regulate adipose tissue mass via its receptors in the ventromedial hypothalamus (VMH). We have therefore tested the hypothesis that stimulation of leptin receptors in the VMH regulates adipocyte number in bone marrow. Results indicate that unilateral twice-daily injections of leptin into the rat VMH for only 4 or 5 days cause a significant reduction in the number of adipocytes in peripheral fat pads and bone marrow and indeed eliminate adipocytes almost entirely from bone marrow of the proximal tibia. Osteoblast surface is not affected with leptin treatment. Apoptosis assays performed on bone marrow samples from control and treated rats have revealed a significant increase in protein concentration of the apoptosis marker caspase-3 with leptin treatment. We conclude that stimulation of leptin receptors in the VMH significantly decreases the adipocyte population in bone marrow, primarily through apoptosis of marrow adipocytes. Elimination of marrow adipocytes via this central pathway may represent a useful strategy for the treatment and prevention of osteoporosis.     

The function of adipocytes in the bone marrow stroma

The fibroblasts and adipocytes of the bone marrow stroma provide the cytokines and extracellular matrix proteins required for the maturation and proliferation of the circulating blood cells. Due to the complexity of the bone marrow as an organ, the normal physiology of these stromal cells is not well understood. In particular, the role of adipocytes in the bone marrow remains controversial. Cloned bone marrow stromal cell lines provide an in vitro model for analysis of the lympho-hematopoietic microenvironment. These cells may be capable of multiple differentiation pathways, assuming the phenotype of adipocytes, chondrocytes, myocytes, and osteocytes in vitro. Characterization of these cell lines and recent in vivo experiments give new insight into the normal physiology of the bone marrow.     

Cannabinoid Receptor Type 1 Protects against Age- Related Osteoporosis by Regulating Osteoblast and Adipocyte Differentiation in Marrow Stromal Cells

Age-related osteoporosis is characterized by reduced bone formation and accumulation of fat in the bone marrow compartment. Here, we report that the type 1 cannabinoid receptor (CB1) regulates this process. Mice with CB1 deficiency (CB1^−/−) had increased peak bone mass due to reduced bone resorption, but developed age-related osteoporosis with reduced bone formation and accumulation of adipocytes in the bone marrow space. Marrow stromal cells from CB1^−/− mice had an enhanced capacity for adipocyte differentiation, a reduced capacity for osteoblast differentiation, and increased expression of phosphorylated CREB (pCREB) and PPARγ. Pharmacological blockade of CB1 receptors stimulated adipocyte differentiation, inhibited osteoblast differentiation, and increased cAMP and pCREB in osteoblast and adipocyte precursors. The CB1 receptor is therefore unique in that it regulates peak bone mass through an effect on osteoclast activity, but protects against age-related bone loss by regulating adipocyte and osteoblast differentiation of bone marrow stromal cells.     

Enhanced accumulation of adipocytes in bone marrow stromal cells in the presence of increased extracellular and intracellular [Ca²⁺

The bone marrow stroma contains osteoblasts and adipocytes that have a common precursor: the pluripotent mesenchymal stem cell found in bone marrow stromal cells (BMSCs). Local bone marrow Ca(2+) levels can reach high concentrations due to bone resorption, which is one of the notable features of the bone marrow stroma. Here, we describe the effects of high [Ca(2+)](o) on the accumulation of adipocytes in the bone marrow stroma. Using primary mouse BMSCs, we evaluated the level of adipocyte accumulation by measuring Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity. High [Ca(2+)](o) enhanced the accumulation of adipocytes following treatment with both insulin and dexamethasone together but not in the absence of this treatment. This enhanced accumulation was the result of both the accelerated proliferation of BMSCs and their differentiation into adipocytes. Using the fura-2 method, we also showed that high [Ca(2+)](o) induces an increase in [Ca(2+)](i). An intracellular Ca(2+) chelator suppressed the enhancement in adipocyte accumulation due to increased [Ca(2+)](o) in BMSCs. These data suggest a new role for extracellular Ca(2+) in the bone marrow stroma: increased [Ca(2+)](o) induces an increase in [Ca(2+)](i) levels, which in turn enhances the accumulation of adipocytes under certain conditions.     

Regulation by cytokines of leptin expression in human bone marrow adipocytes.

Leptin is a hormone secreted by adipocytes. Besides controlling appetite and body weight, it has been suggested that leptin plays a role in inflammation and hemopoiesis. In this study we demonstrate that the pro-inflammatory/hemopoietic cytokines, IL-1beta, IL-6, TNF-alpha, and interferon-gamma, significantly inhibit gene expression and secretion of leptin by bone marrow adipocytes. These findings are in agreement with the data recently obtained from non-medullary adipose tissues. Within the bone marrow environment, leptin regulation by these pleiotropic cytokines could contribute to controlling the proliferation and differentiation of hemopoietic precursors as well as the maturation of stromal cells.     

Melatonin inhibits fatty acid-induced triglyceride accumulation in ROS17/2.8 cells: implications for osteoblast differentiation and osteoporosis

Melatonin is produced not only by the pineal gland but by cells of the bone marrow. Moreover, melatonin is known to promote osteogenic differentiation in several cell line models and in multipotential bone marrow mesenchymal stem cells. Fatty acids have been independently shown to direct such cells to acquire the phenotype and molecular characteristics of adipocytes. To examine the effect of melatonin on intracellular triglyceride accumulation, an indicator of adipogenic differentiation in the rat osteoblast-like ROS17/2.8 cell line, cells were incubated with added oleic acid (100 muM), fixed and stained with Oil Red O. Cellular lipid accumulation was quantitated by an Oil Red O method highly specific for triglycerides and expressed as a triglyceride accumulation index (TGAI, triglyceride per cell). Melatonin in nanomolar concentrations inhibited oleic acid-induced triglyceride accumulation. To identify the mechanism by which melatonin reduces triglyceride accumulation, cells were incubated with the two melatonin receptor antagonists, luzindole and S20928, or forskolin, a stimulator of adenylyl cyclase and cAMP production. These compounds prevented the inhibitory effect of melatonin on triglyceride accumulation, indicating that melatonin acts through known melatonin receptor-mediated mechanisms. In view of the previously demonstrated positive effects of melatonin in promoting osteoblastic differentiation in ROS17/2.8 cells and their reciprocal adipocytic differentiation induced by fatty acids, our observations may serve to relate the known age-related decreases of melatonin production, the shift in the bone marrow toward an adipocytic line of cell development, and the development of osteoporosis during aging.     

New insights into the epigenetics of osteoporosis

Osteoporosis is characterized by reduced bone formation and accumulation of adipocytes in the bone marrow compartment. The decrease in bone mass results from an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The deficiency of bone cells to replace the resorpted bone can be due to a preferential differentiation of bone marrow stromal cells into adipocytes at the expense of osteoblasts. Consequently, the processes that control the differentiation of osteoclasts, osteoblasts and adipocytes play a crucial role in bone metabolism. It is known that epigenetic mechanisms are critical regulator of the differentiation programs for cell fate and moreover are subject to changes during aging. Here, we summarize recent findings on the role of epigenetics in the modulation of mechanisms that may be associated with osteoporosis. In particular, we focus on disturbances in the bone remodeling process described in human studies that address the epigenetic regulation of the osteoblast/adipocyte balance.     

Effect of mammalian growth hormone and prolactin on the growth of hypophysectomized chickens

Body weight gain and shank-toe growth during a 26-day treatment period following hypophysectomy were 55 and 46%, respectively, of control values, but the body weight gain was unaffected and bone growth only slightly reduced when the hypophysectomized chickens were fed a low dose of corticosterone (5 ppm). Bovine growth hormone (0.5 mg GH/kg body wt/day for 18 days) enhanced body weight gain and shank-toe length increase (an estimate of bone growth) by 46 and 33%, respectively, compared to the growth of hypophysectomized chickens receiving only corticosterone. These same endpoints were increased approximately 24% after ovine growth hormone treatment in hypophysectomized chickens not receiving corticosterone. Body weight gain during 18 days of treatment with bovine prolactin (0.5 mg PRL/kg/day) was 27% greater than the value for corticosterone-treated hypophysectomized chickens, but bone growth was unaffected. The mammalian GH preparations increased heart weight of the hypophysectomized chickens (25-29%), but pectoralis muscle weight was unaffected. GH treatment enhanced thymal weights by 71% in corticosterone-treated hypophysectomized chickens, and by 93% in hypophysectomized animals not receiving corticosterone. GH had no significant effect on bursal weights, and PRL had no effect on either of these lymphoid organ weights in corticosterone-treated hypophysectomized chickens. GH increased liver and adipose tissue weights considerably more than the large increases that followed treatment of hypophysectomized chickens with corticosterone alone (69 and 126% greater, respectively), but had no effect on these endpoints in hypophysectomized chickens not receiving corticosterone. PRL also greatly increased liver and adipose tissue weights in corticosterone-treated hypophysectomized chickens (79 and 75%, respectively). These results provide evidence that mammalian GH enhances body weight gain, bone growth, and the growth of several organs in the hypophysectomized chicken. Mammalian PRL increased body weight gain, liver weight, and adipose tissue weight in corticosterone-treated hypophysectomized chickens, but did not influence bone growth or the weights of the heart, pectoralis, thymi, or bursa.     

Propranolol attenuates calorie restriction- and high calorie diet-induced bone marrow adiposity

We investigated the effects of β-adrenergic activation on bone marrow adiposity and on adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). C57BL/6 mice were subjected to a control (CON), high calorie (HIGH) or low calorie (LOW) diet for 12 weeks. In each group, mice were treated with vehicle (VEH) or propranolol. The number of adipocytes per area bone marrow was increased in LOWVEH and HIGHVEH mice compared with CONVEH mice, which was attenuated by propranolol. Isoproterenol increased lipid droplet accumulation and adipogenic marker gene expression in 3T3-L1 preadipocytes and mouse BMSCs, which were blocked by propranolol. Conditioned medium obtained from MC3T3-E1 osteoblasts suppressed adipogenic differentiation of 3T3-L1 cells, which was significantly attenuated by treatment of MC3T3-E1 cells with isoproterenol. These data suggest that β-adrenergic activation enhances bone marrow adipogenesis via direct stimulation of BMSCs adipogenesis and indirect inhibition of osteoblast anti-adipogenic potential. [BMB Reports 2014; 47(10): 587-592]     

Growth Hormone Treatment of Hypophysectomized Rats Increases Catecholamine-induced Lipolysis and the Number of β-Adrenergic Receptors in Adipocytes: No Differences in the Effects of Growth Hormone on Different Fat Depots

Growth hormone (GH) has a lipolytic effect in adipose tissue but this effect may differ in adipose tissue from various fat depots. This latter possibility was investigated in the present study, in which the effects of GH in vivo on catecholamine-induced lipolysis and the number of β-adrenergic receptors in isolated adipocytes from different fat depots of hypophysectomized rats were investigated. Female and male Sprague-Dawley rats were hypophysectomized or sham-operated at 45 days of age. One week after the operation, hormonal replacement therapy with L-thyroxine and hydrocortisone acetate was given. In addition, groups of rats were treated with GH (1.33 mg/kg per day, given as two daily subcutaneous injections). After 1 week of hormonal treatment, adipocytes were isolated from the parametrial, epididymal and inguinal fat pads, and glycerol release after catecholamine-stimulation and ¹²⁵I-cyanopindolol binding were measured. Hypophysectomy resulted in a marked decrease in the lipolytic response to catecholamines. GH treatment significantly increased catecholamine-induced lipolysis with similar effects in adipocytes from parametrial or epididymal and inguinal fat depots in both female and male rats. There were no differences between norepinephrine compared with isoproterenol-induced responses. ¹²⁵I-cyanopindolol binding was reduced after hypophysectomy and normalized by GH treatment, without differences between parametrial and inguinal adipose tissue regions. We conclude that the lipolytic effects of GH in the rat may partly be mediated by a stimulatory effect on β-adrenergic receptors in adipocytes. In addition, GH exerted similar effect on catecholamine-induced lipolysis and β-adrenergic receptors in adipocytes from parametrial, epididymal and inguinal fat depots.     

Effects of hypophysectomy and acute growth hormone treatment upon glucose metabolism in adipose tissues and isolated adipocytes of rats.

Glucose oxidation and incorporation into lipid were measured in epididymal adipose tissues and isolated adipose cells of normal and hypophysectomized rats in an effort to determine whether the acute hypoglycemic effect of a systemic growth hormone (GH) injection was related to alterations in the glucose metabolism of adipose tissue. The rats were fed rat chow or a high sucrose diet and received 100 mug GH intraperitoneally 30 minutes or three and one-half hours before sacrifice. Hypophysectomized rats showed a lower plasma glucose as compared with normal rats on both diets. Thirty minutes after a GH injection there was a further decrease of the plasma glucose which, however, was not present in those rats receiving GH three and one-half hours before sacrifice. Adipose tissues from hypophysectomized rats fed the high sucrose diet showed a blunted insulin sensitivity as compared with normal rats on a similar diet. The insulin sensitivity of these tissues was further decreased 30 minutes after a GH injection. Basal glucose metabolism of isolated adipocytes from hypophysectomized rats, as compared with normal rats, was depressed if they were fed rat chow, was at normal levels if they were fed the high sucrose diet and was increased if they were fed the sucrose diet and received triiodothyronine and cortisone supplements. No manipulations of diet or hormonal treatments made the isolated adipocyte from hypophysectomized rats sensitive to insulin either 30 minutes or three and one-half hours after a GH injection. Since basal glucose utilization is not enhanced by GH injection and both the blunted insulin sensitivity of adipose tissue and the absent insulin sensitivity of adipopocytes would be expected to produce hyperglycemia rather than hypoglycemia, it is concluded that immediate systemic effects of a GH injection on carbohydrate metabolism are not related to changes in glucose metabolism of the peripheral adipose tissues.     

The interplay of osteogenesis and hematopoiesis: expression of a constitutively active PTH/PTHrP receptor in osteogenic cells perturbs the establishment of hematopoiesis in bone and of skeletal stem cells in the bone marrow

The ontogeny of bone marrow and its stromal compartment, which is generated from skeletal stem/progenitor cells, was investigated in vivo and ex vivo in mice expressing constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrP; caPPR) under the control of the 2.3-kb bone-specific mouse Col1A1 promoter/enhancer. The transgene promoted increased bone formation within prospective marrow space, but delayed the transition from bone to bone marrow during growth, the formation of marrow cavities, and the appearance of stromal cell types such as marrow adipocytes and cells supporting hematopoiesis. This phenotype resolved spontaneously over time, leading to the establishment of marrow containing a greatly reduced number of clonogenic stromal cells. Proliferative osteoprogenitors, but not multipotent skeletal stem cells (mesenchymal stem cells), capable of generating a complete heterotopic bone organ upon in vivo transplantation were assayable in the bone marrow of caPPR mice. Thus, PTH/PTHrP signaling is a major regulator of the ontogeny of the bone marrow and its stromal tissue, and of the skeletal stem cell compartment.     

Stability of bone marrow stromal cells to low temperatures during differentiation

On the bases of earlier conducted research about the stability of heterogeneous population of keratinocytes to low temperatures according to their stages of differentiation this experiment' studies in vitro the stability to low temperatures of rat bone marrow stromal cells before and after their adipocyte and osteocyte differentiation. Results show that bone marrow stromal cells after their differentiation into either adipocytes or octeocytes became least stable to low temperatures. Findings may serve as foundation for further studies that may explain the changes of processes and mechanisms that play a major role in BMSC stability to low temperatures according to their stage of differentiation.     

Induction of growth hormone release by glycyrrhizae radix on rat

Induction of growth hormone (GH) by Glycyrrhizae Radix (GR), one of the most popular herbal medicine, and its major ingredients were studied in rat pituitary cells in vitro and in vivo assay. The MeOH extract and the n-hexane (HX) fraction of GR induced rat GH (rGH) release up to 1.89 times (0.34 +/- 0.04 nM) and 4.59 times (0.83 +/- 0.03 nM), compared to the basal level (p < 0.05). Among many ingredients isolated and purified from GR both glycyrrhetinic acid and glycyrrhizin induced significantly rGH release compared to the control (p < 0.05). After an intravenous injection of rat growth hormone releasing hormone (rGHRH) (10 microg/kg) as positive control, in SD rats, Tmax of plasma rGH level was 10 min, C(max) was 3.84 +/- 0.01 nM (n = 3), and enhanced plasma rGH level returned to the baseline in 90 min. Both AUC(0-90) (area under the curve) of plasma rGH level after HX fraction and that after rGHRH administration were increased significantly from the basal level, respectively (p < 0.01). In conclusions, HX fraction is the most active fraction of MeOH extract of GR in rGH induction.