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Keywords:

  • leptin;
  • leptin receptor;
  • osteoblasts;
  • osteosarcomas

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

The adipose hormone leptin and its receptor are important for regulation of food intake and energy metabolism. Leptin also is involved in the growth of different tissues. In this study, we show the expression of leptin in primary cultures of normal human osteoblasts (hOBs) as evidenced by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. Release of leptin into the medium also was found. Leptin was not detected in commercially available hOBs (NHOst) or in three different human monoclonal osteosarcoma cell lines. Leptin expression was observed in OBs in the mineralization and/or the osteocyte transition period but not during the matrix maturation period. Furthermore, hOBs and osteosarcoma cell lines expressed the long signal-transducing form of the leptin receptor (OB-Rb) as shown by RT-PCR. We observed no significant changes in leptin or OB-Rb genes in hOBs after incubation with recombinant leptin, indicating no autoregulation of the leptin expression. Incubation of both hOBs entering the mineralization phase and osteosarcoma cell lines with recombinant leptin markedly increased the number of mineralized nodules as shown by alizarin S staining. These findings indicate that leptin may be of importance for osteoblastic cell growth and bone mineralization.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

LEAN BODY status is an important risk factor for low bone mineral density (BMD), as well as for fractures in the hip, wrist, and vertebrae.(1, 2) A protective effect of overweight/obesity on BMD has been ascribed to high body mass promoting increased mechanical load on weight-bearing bones. In postmenopausal women, the main source of estrogen is produced via the conversion of androstenedione to estrone in adipose tissue. Consequently, lean women have less capacity to produce estrogen.(3) A third explanation for the association between body mass and osteoporosis may be related to the plasma protein leptin originating from the obese gene.(4) It has been shown in the natural leptin knockout model (ob/ob mice) that leptin administration increases BMD, as well as limb length.(5) Recently, it was shown that leptin induced differentiation of human bone marrow stromal cells into osteoblasts (OBs), while impeding the maturation of adipocytes.(6) All these results suggest a role for leptin in bone metabolism.

Expression of leptin messenger RNA (mRNA) has been associated primarily with adipocytes,(7–9) but it also has been found in human placental syncytiotrophoblasts,(10, 11) gastric epithelium,(12) activated rat hepatic stellate cells,(13) and activated murine muscle cells.(14) Moreover, it also has been shown that leptin and the leptin receptor are expressed in murine fetal cartilage and bone.(15) In this report, we show leptin expression in primary cultures of human OBs (hOBs) and the release into the cell media. We also show that hOBs and osteosarcoma cells express the long signal-transducing form of the leptin receptor (OB-Rb) and that long-term incubation of recombinant leptin facilitates mineralization. These results indicate that leptin may act via autocrine and endocrine mechanisms in the regulation of bone metabolism.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Cell cultures

The isolation of hOB cells was performed essentially as described by Robey and Termine.(16) Trabecular bone specimens (caput femoris and iliac crest) were obtained from osteoarthritic patients without malignant disease undergoing hip surgery. Soft connective tissue and periosteal and cortical bone were removed, and the remaining trabecular bone was minced and extensively washed with phosphate-buffered saline (PBS) to remove bone marrow cells. Then, the bone fragments, approximately 3 mm3, were digested at 37°C with 1 mg/ml type H bacterial collagenase (Sigma Chemical Co., St. Louis, MO, USA) in Dulbecco's modified Eagle's medium/F12 (DMEM/F12; Gibco, Paisley, UK). After 2 h, the released cells were discarded and the remaining bone fragments were washed extensively with DMEM/F12 containing 10% fetal calf serum (FCS). The fragments were seeded into 25-cm2 tissue culture flasks (Costar, Cambridge, MA, USA) and incubated at 37°C in a humidified atmosphere with 5% CO2. The culture medium DMEM/F12 was supplemented with 10% FCS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 1 mM pyruvate, and 2 mM glutamine. The medium was changed weekly until confluent cell monolayer was obtained after 4-6 weeks. Confluent cells (10,000 cells/cm2) were detached with trypsin (2.5%) and EDTA (0.02%) and subcultured (dilution 1:4) further with an in vitro life span of 35-44 days.

Bone cultures from four different donors were used for the experiments in this study. In most of the experiments with primary hOBs, the cells were cultured for 35-44 days. For the immunodetection Oil Red-O staining presented hOB was trypsinized and recultured on slides 3 days before staining. Trypsinization and subculturing of these cells within this period had no effect on the results presented here.

In some experiments cells also were tested for leptin expression after 21 days. As determined by an alkaline phosphatase activity (ALP) staining kit from Sigma Chemical Co. (85L-2), 50-60% of the cells in the bone culture stained positive for ALP after 35 days in culture and, thus, represents differentiated OBs. This is in accordance with the original report describing isolation and characterization of hOB cells.(16) Commercially available hOBs (NHOst cell system; Clonetics, San Diego, CA, USA) were grown in Osteoblast Growth Media (OGM) (Clonetics). Monoclonal lines of human osteosarcoma OHS,(17) KPDXM(18) (established from tumors by Dr. Ø.S. Bruland, Oslo, Norway), and 788T (kindly supplied by Dr. E.J. Embleton, Nottingham, UK) were grown in RPMI 1640 (Gibco) with 10% FCS, 50 IU/ml penicillin, and 50 μg/ml streptomycin (Gibco). Medium was changed every 2-3 days and always 24 h before harvesting. Murine 3T3-L1 (CL-173) preadipocytes (American Type Culture Collection, Manassas, VA, USA) were grown in DMEM fortified with FCS and antibiotics as described previously. Differentiation into mature adipocytes was accomplished by incubating the cells with insulin, dexamethasone, and 3-isobutyl-1-methylxanthine (IBMX) as described previously.(19)

mRNA isolation and semiquantitative reverse transcriptase-polymerase chain reaction

OBs, osteosarcoma cells, and 3T3-L1 adipocytes were lysed in lysis/binding buffer (100 mM Tris-HCl, pH 8.0, 500 mM LiCl, 10 mM EDTA, pH 8.0, 0.5 mM dithiothreitol [DTT], and 1% sodium dodecyl sulfate [SDS]). Human subcutaneous clamp-frozen adipose tissue (250 mg) was crushed in liquid nitrogen, lysed as mentioned previously, and homogenized using an Ultra-Turrax (The Vertis Co., Gardiner, NY, USA).

mRNA was isolated using magnetic beads [oligo (dT)25] as described by the manufacturer (Dynal AS, Oslo, Norway). For analysis of leptin and leptin receptor mRNAs, beads containing mRNA were resuspended in 10 mM Tris-HCl, pH 8.0, and stored at −70°C until use. The GeneAmp EZ rTth RNA PCR kit (Perkin Elmer, Applied Biosystems, Foster City, CA, USA) was used for the reverse transcriptase-polymerase chain reaction (RT-PCR) and 2 μCi [32P]deoxycytosine triphosphate (dCTP) was added for each reaction. Temperature cycles were as follows: 60°C for 30 minutes, 94°C for 1 minute followed by 25-45 cycles at 94°C for 30 s, and 60°C for 1.5 minutes. At the end, the samples were incubated at 60°C for 7 minutes. Ten microliters of the reaction mixture was separated on 2% agarose gels and stained with ethidium bromide, excised, and counted for 1 minute in a liquid scintillation counter (Packard 1900 TR; Packard, Chicago, IL, USA). The detection of leptin mRNA was based on 40 cycles, whereas the comparison between relative levels of leptin in OBs, BeWo, and adipocytes was performed after 34 cycles and was within the linear area of PCR amplification.

For analysis of OB cell markers, cells were pelleted at 700g, washed in PBS and frozen at −80°C in aliquots of 4 × 106 cells. Subsequently frozen cells were lysed in lysis/binding buffer and mRNA was isolated using Dynabead (Dynal AS). After annealing and washing the beads, mRNA was eluted in 40 μl diethylpyrocarbonate (DEPC) dH20 at 65°C. The mRNA-containing solution was applied directly to obtain a first-strand complementary DNA (cDNA) using the Pharmacia Biotech kit (with random hexamer primers and Moloney murine leukemia virus RT; Pharmacia Biotech, Uppsala, Sweden). Incubation conditions were 37°C for 60 minutes. The PCR-amplification reaction contained 10 μl of the cDNA mixture, 15 pmol of sense and antisense primers, 2 μCi of [32P]dCTP (3000 Ci/mmol), 2.5 mM Mg2+, and 2.5 U of Taq polymerase. The cycling profile was as follows: denaturing at 94°C for 5 minutes followed by 20-40 cycles of annealing at 59°C for 30 s, primer extension at 72°C for 45 s, and denaturing at 94°C for 30 s. Finally, one cycle for 3 minutes of extension completed the reaction. Ten microliters of the reaction mixture was applied on 6% polyacrylamide gel electrophoresis (PAGE; Novex, Invitrogen, Carlsbad, CA, USA) and stained with ethidium bromide, excised, and counted for 1 minute as described previously. The primers were selected by using Primer Analysis, Oligo version 4.0 software (National Biosciences, Plymouth, MN, USA) and designed to give optimal annealing at 59°C.

Relative abundance of phenotype marker mRNAs was calculated as glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA ratios. Oligonucleotide sequences of sense and antisense primers were as follows: collagen (α1), estimated product size 306 base pairs (bp), 5′-GCAAGAACCCCAAGGACAAGAG-3′ and 5′-TCGTGCAGCCATCGACAGTGAC-3′; osteocalcin (bone GLA protein), estimated product size 257 bp, 5′-GGCAGCAGGTAGTGAAGAGAC-3′ and 5′-GGCAAGGGGAAGAGGAAAGAAG-3′; ALP, estimated product size 341 bp, 5′-CACGGGCACCATGAAGGAAAAG-3′ and 5′-TGGCGCAGGGGCACAGGAGACT-3′; parathyroid hormone (PTH) receptor, estimated product size 284 bp, 5′-CTGGACACTGGCACTGGACTTC-3′ and 5′-GGCCTGAGCAGGAGCCGTTGAG-3′; hormone-sensitive lipase (HSL),estimated product size 320 bp, 5′-AGGTGTTCGGGAACAGGCACTGG-3′ and 5′-CGCCCTCAAAGAAGAGCACTCCT-3′; human leptin, estimated product size 197 bp, 5′-GGCTTTGGCCCTATCTTTTC-3′ and 5′-GGATAAGGTCAGGATGGGGT-3′; the common extracellular region of the human leptin receptor (OB-Rex), estimated product size 565 bp, 5′-TCCCATATCTGAGCCCAAAG-3′and 5′-CATCAGGGGCTTCCAAAGTA-3′; Signaling form of leptin receptor (OB-Rb), product size 417 bp, 5′-GCCAGAGACAACCCTTTGTTAAA-3′ and 5′-TGGAGAACTCTGATGTCCGTGAA-3′; G3PDH, product size 452 bp: 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′.

Quantitation of leptin in culture media

The proteins in cell culture media incubated with OBs were concentrated 10- to 20-fold by freeze-drying and dissolved in PBS. Culture medium containing 10% FCS was treated similarly and used as negative control. Concentration of leptin was measured by a competitive radioimmunoassay (Linco Research, St. Charles, MO, USA) with recombinant125I-leptin as tracer.(20) The concentration of leptin found in culture media with 10% FCS (0.005 ± 0.007 ng leptin/ml media) was subtracted from the level of leptin obtained from OBs after 24-h incubation with an intra-assay variation of 2.1%.

Immunocytochemistry

Primary hOBs (isolated hOB cells) and commercially available hOBs (NHOst) were cultured on chamber slides (Lab-Tek Flaskette; Nunc International, Naperville, IL, USA), air-dried, fixed for 10 minutes in buffered 4% formaldehyde at room temperature, rinsed, and incubated in 3% H2O2 for 10 minutes to block endogenous peroxidase activity. Immunocytochemistry was performed with the avidin-biotin peroxidase complex (ABC) technique (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA) and tyramide signal amplification (TSA Indirect; NEN Life Science Products, Boston, MA, USA) according to manufacturers' instruction with rinsing in cold PBS containing 0.25% Triton X-100 (Calbiochem, La Jolla, CA, USA) between each step. The antisera (rabbit anti-human leptin; PeproTech EC Ltd., London, UK) and rabbit immunoglobulin fraction (normal; Dako, Glosrup, Denmark) were diluted 1:500 in PBS containing 0.25% Triton X-100 and 0.25% bovine serum albumin (BSA; Sigma Chemical Co.) and incubated overnight at 4°C. 3-Amino-9-ethylcarbazole (AEC; Vector Laboratories) was used as chromogen, and the slides were counterstained with Mayer's hematoxylin for 2 s.

Oil Red-O lipid staining

Lipid staining was done with the Oil Red-O method.(21) Adipocytes (3T3-L1 [CL-173]; American Type Culture Collection) and OBs (isolated hOB) were cultured on chamber slides (Lab-Tek Flaskette). The staining solution was prepared by dissolving 0.5 g Oil Red-O powder (Sigma Chemical Co.) in 100 ml of isopropanol, followed by 3:2 dilution with distilled water. Then, the solution was allowed to stand for 10 minutes before it was filtered through a 5-μm nylon mesh. The cells were washed in PBS, fixed as described previously, and stained with Oil Red-O for approximately 1 h. All samples were washed with distilled water and counterstained with Mayer's hematoxylin for 2 s.

Effect of recombinant leptin on the expression of leptin and leptin receptor

OBs were incubated with 1 ng/ml and 100 ng/ml recombinant leptin (BIOMOL Research Laboratories, Inc., Plymouth, PA, USA) in media containing 10% FCS. Media were changed every 24 h. The expression of leptin and leptin receptor (OB-Rb) mRNA was measured after 6, 24, 48, and 72 h of incubation and compared with the expression in cells incubated without leptin.

OB mineralization

Cells were grown in RPMI 1640 medium with L-glutamine containing standard levels of penicillin, streptomycin, fungizone, and anti-pleuropneumonia-like organisms (PPLO)-agent. In 35-44 days of cultured primary OBs and confluent osteosarcoma cells, the medium was fortified with β-glycerophosphate (10 mM) and ascorbic acid (50 μg/ml) and incubated for another 35 days with or without leptin (100 ng/ml). The cells were rinsed three times with PBS and fixed with 95% methanol for 30 minutes. Subsequently, the cells were stained with 1% alizarin red S at pH 6.4 for 5 minutes and washed with distilled water as previously described.(22, 23) Images of the mineralized nodules were obtained with a Zeiss standard microscope (Carl Zeiss, Oberkochen, Germany) equipped with a CCD video camera (Hamamatsu C3077; Hamamatsu, Hamamatsu, Japan) and stored on a Macintosh computer running National Institutes of Health (NIH) image software. Percent surface covered by mineral was estimated using a Zeiss I integrating eyepiece with seven parallel lines. Four hundred intersections between lines and mineralized surface were counted.(24)

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Cell culture and characterization of primary hOBs

The primary cultures of hOB and osteosarcoma cells were characterized by the expression of OB phenotypic markers like collagen (α1), ALP, osteocalcin, and PTH receptor as well as the adipocyte marker HSL, relative to that of G3PDH. The phenotypic OB genes are known to have temporal expression during 35 days of cell culture.(9) Peak levels of collagen are associated with cell proliferation. ALP activity is found in the matrix maturation period, and osteocalcin expression is related to the mineralization phase reflecting calcium deposition.(9) The OB phenotype was assessed by comparison with white adipose tissue (Table 1). After 35 days of culture hOB, 50-60% of the cells stained positive for ALP activity and expression of collagen and osteocalcin was higher than in osteosarcoma cells. HSL expression was virtually absent in the OBs, indicating that they did not display a characteristic feature of adipocytes. In contrast, white adipose tissue expressed high levels of HSL and markers of the OB phenotype were virtually nonexistent or absent.

Table Table 1.. Characterization of Cells Based on the Relative Amounts of mRNA
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Differentiation toward or contamination with adipocytes expressing leptin may be recognized by the appearance of lipid droplets in the cultured cells and can be monitored by Oil Red-O staining. There were no lipid droplets in cultured primary hOB cells (Fig. 1A). This observation, along with little or no detection of HSL, indicated the absence of mature adipocytes in the primary culture. Murine adipocytes (3T3-L1 cells) served as positive controls and showed extensive accumulation of Oil Red-O stained lipid droplets (Fig. 1B).

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Figure FIG. 1.. Visualization of lipid droplets using Oil Red-O staining. (A) Primary hOBs cultured for 35-44 days showed no staining. (B) The red lipid droplets were clearly visible in mature mouse adipocytes (3T3-LI) used as control. We did not observe any contamination with mature adipocytes or adipocyte-like cells in the hOB cell culture. Our results from a single individual are representative of findings from 4 separate individuals. One hundred micrometers is indicated in upper left corner.

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Leptin and leptin receptor expression

Leptin mRNA was expressed in normal hOBs cultured for 35 days. The expression of leptin was evaluated by semiquantitative RT-PCR to be in the same range as in human trophoblasts (BeWo) (Fig. 2A) and white adipose tissue (Fig. 2B). After only 21 days of culture, no leptin mRNA was observed (data not shown). Eighty to one hundred percent of these hOB cells expressed ALP activity characteristic of the matrix maturation phase of the OB development. No leptin mRNA was detected in commercially available NHOst cells or in immortalized osteosarcoma cell lines (788T, KPDXM, and OHS). By using primers for the common extracellular region of human leptin receptor (OB-Rex) and the long intracellular signal-transducing form (OB-Rb), expression of the leptin receptor mRNA was shown in OBs as well as osteosarcoma cell lines (Fig. 2A). Primary cultures of hOBs displayed more leptin receptor mRNA than NHOst and osteosarcoma cells (Fig. 2A).

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Figure FIG. 2.. Expression of leptin and leptin receptor mRNA in primary hOBs from (A) one individual (hOB 1), NHOst cells, and osteosarcoma cell lines (788T, KPDXM, and OHS) compared with the expression in human trophoblasts (BeWo). (B) Another individual (hOB 2) and white adipose tissue (WAT). The OBs (hOB 1 and hOB 2) were cultured for 35-44 days, and the presented data were representative of findings from 4 individuals. mRNAs from the cultured cells and human subcutaneous adipose tissue were isolated by employing oligo-dT-linked magnetic beads and the corresponding cDNAs were achieved by RT-PCR analyses.

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Expression of leptin protein was shown in 35- to 44-day-old cultured hOB by immunostaining (Fig. 3A), and NHOst cells showed no immunoreactivity (Fig. 3B). Irrelevant antiserum (normal rabbit immunoglobulin fraction) was used as negative control. Along with the detection of leptin mRNA and leptin immunoreactivity in hOB cells, significant amounts of leptin could be found in the culture medium supernatant harvested after 24-h incubation in control medium (0.14 ± 0.02 ng leptin/ml).

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Figure FIG. 3.. Immunostaining of leptin in hOBs. Leptin immunoreactivity was observed in the (A) cytoplasm of primary hOBs cultured 35-44 days and (B) no staining was found in commercially available hOBs (NHOst). The presented result from a single individual is representative of findings from 4 individuals. The cells presented are trypsinized and subcultured on slides 3 days before staining. One hundred micrometers is indicated in upper left corner.

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Effect of recombinant leptin on the cellular expression of leptin and leptin receptor

No significant changes were observed in the expression of leptin or leptin receptor (OB-Rb) gene in OBs after incubation with moderate (1 ng/ml) or high (100 ng/ml) concentrations of recombinant leptin over a period ranging from 6 to 72 h (results not shown).

Effect of leptin on mineralization in OBs and osteosarcoma cell lines

Confluent cells in monolayers were incubated in standard medium fortified with β-glycerol-phosphate (10 mM) and ascorbic acid (50 μg/ml). Mineralized nodules were photographed (Figs. 4A and 4B) and the area covered by mineral crystals was scored by microscopy (Fig. 4C). Leptin (100 ng/ml) enhanced mineralization in primary hOB more than 6-fold (from 8% to 42%). The same phenomenon was observed in osteosarcoma cells where mineralization increased 2.5-, 4-, and 2-fold in 788T, KPDXM, and OHS cells, respectively.

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Figure FIG. 4.. Mineralization of hOBs cultured for 35-44 days and osteosarcoma cell lines was estimated after another 35 days of incubation (A) without or (B) with leptin (100 ng/ml). Panels A and B illustrate the microscopic view of the mineralization nodules in OBs. (C) The calculated percent surface area covered by mineral crystals in OBs and osteosarcoma cell lines (788T, KPDXM, and OHS). Confluent cells in monolayer cultures were incubated in standard medium with β-glycerophosphate and ascorbic acid. Subsequently, the cultures were washed in PBS, fixed in 95% methanol, and stained with alizarin red S.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

For the first time we have shown the transcription, translation, and secretion of leptin from primary hOBs. The cells isolated from bone explants were characterized as OBs based on the expression of phenotypic markers and ALP activity, and they did not exhibit Oil Red-O staining. We observed no signs of adipocyte-like differentiation or contamination with mature adipocytes within the isolated hOB cells. Leptin has been found previously in various fetal bone tissues,(25) whereas our results indicate an abundant expression of leptin in OBs isolated from adult bone tissue. However, it appears that the expression of leptin may be limited to the mineralization and/or osteocyte transition period of OB differentiation.(9) Our data are single time-point observations and we cannot exclude the possibility of nonosteogenic cells in the primary culture.

Primary OBs and different osteosarcoma cell lines expressed the long form of the leptin receptor (OB-Rb), suggesting a role for leptin in the direct regulation of human bone metabolism. Our present data were confirmed recently in an abstract at the 22nd American Society for Bone and Mineral Research (ASBMR) Annual Meeting by Bassilana et al., showing that human mesenchymal stem cells undergoing osteoblastic differentiation express leptin and OB-Rb,(26) and by Steppan et al.,(27) identifying the long form of OB-R on human primary OBs by Western blotting. The study by Ducy et al. is in contradiction to our data related to the expression of leptin and its receptor in OBs.(28) This could be caused by species difference or the fact that we have higher sensitivity for detection of expression.

Tissue-specific autoregulation of leptin expression has been shown recently in adipocytes and muscle cells.(29) We observed no significant changes in leptin or OB-Rb genes in OBs after incubation with recombinant leptin, indicating no autoregulation of leptin in primary cultures of hOB.

Exogenously added leptin stimulated the production of mineralization nodules in hOBs entering the mineralization and/or osteocyte transition phase and osteosarcoma cell cultures in vitro. The presence of a functional OB-Rb in hOBs was confirmed by Bassilana et al. showing that leptin induced STAT3 activation and c-fos expression.(26) Several isoforms of the leptin receptor, including OB-Rb, also have been shown in murine osteoblastic cell lines.(30) Moreover, it has been shown that leptin stimulates cortical bone formation in young obese mice (ob/ob)(31) as well as increases the number of mineralized nodules in cultures of neonatal rat calvarial cells.(8) However, Ducy et al. found that exogenous leptin inhibited bone formation in ob/ob and wild-type mice,(28) whereas Steppan et al. showed an increased femoral length, mineral content, bone area, and bone density in ob/ob mice after leptin administration.(27) Contrary to our observation, Ducy et al.(28) could not detect expression of leptin in murine OBs using Northern blot analysis. We have used a more sensitive RT-PCR technique in the detection of gene expression and verified the translation of the transcript using antibodies to human leptin. It is possible that there are species differences between mice and humans for the expression of both leptin and its receptor, as well as for the effect of leptin on bone metabolism. However, one must be cautious in hypothesizing on the role of leptin in bone metabolism, because in vitro observations in humans(26) and rodents(30, 31) are in conflict with one in vivo study in mice(28) while in accordance with another.(27)

The differentiation of the osteoprogenitor cell is regulated by the concerted action of several growth factors, morphogenic proteins (bone morphogenic protein [BMP]-2, -4, and -7), and cytokines produced in the bone microenvironment as well as systemic hormones like PTH, 17β-estradiol, and calcitriol.(9) BMP-2 enhances gene transcription of the OB marker collagen-1α, ALP, and osteocalcin in bone marrow cells.(7) Leptin mediates at least some of its biological effect via its receptor belonging to the class I cytokine receptor family.(11) OBs also respond to interleukins by differentiation into osteocytes.(10) Cytokines as well as leptin are produced by OBs and may interact with their specific receptors executing regulation of bone growth. Thus, the effect of leptin on bone cells may resemble that of interleukins; that is, leptin might stimulate OB differentiation and inhibit the differentiation of bone marrow stromal cells into adipocytes.(12)

The reason why leptin is not expressed in undifferentiated OBs and immortalized osteosarcoma cells is unknown. Leptin expression might be an indicator of differentiation and might be induced when OBs enter the mineralization and/or osteocyte transition phase of cell development. During malignant transformation, expression of genes like leptin may be lost because of dedifferentiation or a general genetic instability of cancer cells.(13) Accumulation of mutations may lead to defective protein synthesis and decrease or loss of cell markers.(13)

In one study, serum leptin and periosteal bone expansion were positively related during growth among 8- to 13-year-old girls,(14) whereas there was no association between serum leptin concentration and bone remodeling markers in other studies,(15, 32–34) This can be interpreted as if locally produced leptin may be more important than circulating leptin in regulation of bone metabolism. In addition to the expression of leptin and its receptor in primary cultures of hOBs, we observed increased mineralization by incubation with recombinant leptin, which is in accordance with previously presented data in an abstract from 1997.(8) Although several reports find no direct association between plasma leptin concentration and markers of bone growth, there still may be an important link between adipose tissue and bone metabolism via common signal cascades.

In conclusion, we have for the first time shown expression of leptin and its receptor in normal hOBs. Furthermore, we have shown that leptin stimulates bone mineralization in mature cultured OBs or preosteocytes. Our present findings provide a possible explanation for the observed relationship between energy balance, adipose mass, and bone metabolism.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
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