Leptin, the product of the obese gene, is a circulating hormone secreted primarily from adipocytes. Several results suggest that leptin is important mediators of bone metabolism. The present study was undertaken to determine the effects of leptin on anti-osteoclastogenesis using murine precursors cu...
Leptin, the product of the obese gene, is a circulating hormone secreted primarily from adipocytes. Several results suggest that leptin is important mediators of bone metabolism. The present study was undertaken to determine the effects of leptin on anti-osteoclastogenesis using murine precursors cultured on Ca-P coated plates and on the production of osteoprotegerin (OPG) in osteoblastic cells. Additionally, this study examined the possible involvement of prostaglandin $E_2\;(PGE_2)$/protein kinase C (PKC)-mediated signals on the effect of leptin on anti-osteoclastogenesis to various culture systems of osteoclast precursors. Osteoclast generation was determined by counting tartrate-resistant acid phosphatase positive [TRAP (+)] multinucleated cells (MNCs). Osteoclastic activity was determined by measuring area of resorption pits formed by osteoclasts on Ca-P coated plate. The number of 1,25-dihydroxycholecalciferol $(1,25[OH]_2D_3)$- or $PGE_2$-induced TRAP (+) MNCs in the mouse bone marrow cell culture decreased significantly after treatment with leptin. The number of receptor activator of NF-kB ligand (RANKL)-induced TRAP (+) MNCs in M-CSF dependent bone marrow macrophage (MDBM) cell or RAW264.7 cell culture decreased significantly with leptin treatment. Indomethacin inhibited osteoclast generation induced by $1,25[OH]_2D_3$ and dexamethasone, however, no significant differences were found in the leptin treated group when compared to the corresponding indomethacin group. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, inhibited osteoclast generation induced by $1,25[OH]_2D_3$. The number of TRAP (+) MNCs decreased significantly with treatment by PMA at concentrations of 0.01 and $0.1{\mu}M$ in culture. Leptin inhibited PMA-mediated osteoclast generation. Isoquinoline-5-sulfonic 2-methyl-1-piperazide dihydrochloride (H7) had no effect on osteoclast generation induced by $1,25[OH]_2D_3$. Cell culture treatment with leptin resulted in no significant differences in osteoclast generation compared to the corresponding H7 group. Indomethacin showed no significant effect on TRAP (+) MNCs formation from the RAW264.7 cell line. PMA inhibited TRAP (+) MNCs formation induced by RANKL in the RAW264.7 cell culture. H7 had no effect on osteoclast generation from the RAW264.7 cell line. There was no difference compared with the corresponding control group after treatment with leptin. $1,25[OH]_2D_3$- or $PGE_2$-induced osteoclastic activity decreased significantly with leptin treatment at a concentration of 100 ng/ml in mouse bone marrow cell culture. Indomethacin, PMA, and H7 significantly inhibited osteoclastic activity induced by $1,25[OH]_2D_3$ in mouse bone marrow cell culture. No significant differences were found between the leptin treated group and the corresponding control group. The secretion of OPG, a substance known to inhibit osteoclast formation, was detected from the osteoblasts. Treatment by leptin resulted in significant increases in OPG secretion by osteoblastic cells. Taken these results, leptin may be an important regulatory cytokines within the bone marrow microenvironment.
Leptin, the product of the obese gene, is a circulating hormone secreted primarily from adipocytes. Several results suggest that leptin is important mediators of bone metabolism. The present study was undertaken to determine the effects of leptin on anti-osteoclastogenesis using murine precursors cultured on Ca-P coated plates and on the production of osteoprotegerin (OPG) in osteoblastic cells. Additionally, this study examined the possible involvement of prostaglandin $E_2\;(PGE_2)$/protein kinase C (PKC)-mediated signals on the effect of leptin on anti-osteoclastogenesis to various culture systems of osteoclast precursors. Osteoclast generation was determined by counting tartrate-resistant acid phosphatase positive [TRAP (+)] multinucleated cells (MNCs). Osteoclastic activity was determined by measuring area of resorption pits formed by osteoclasts on Ca-P coated plate. The number of 1,25-dihydroxycholecalciferol $(1,25[OH]_2D_3)$- or $PGE_2$-induced TRAP (+) MNCs in the mouse bone marrow cell culture decreased significantly after treatment with leptin. The number of receptor activator of NF-kB ligand (RANKL)-induced TRAP (+) MNCs in M-CSF dependent bone marrow macrophage (MDBM) cell or RAW264.7 cell culture decreased significantly with leptin treatment. Indomethacin inhibited osteoclast generation induced by $1,25[OH]_2D_3$ and dexamethasone, however, no significant differences were found in the leptin treated group when compared to the corresponding indomethacin group. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, inhibited osteoclast generation induced by $1,25[OH]_2D_3$. The number of TRAP (+) MNCs decreased significantly with treatment by PMA at concentrations of 0.01 and $0.1{\mu}M$ in culture. Leptin inhibited PMA-mediated osteoclast generation. Isoquinoline-5-sulfonic 2-methyl-1-piperazide dihydrochloride (H7) had no effect on osteoclast generation induced by $1,25[OH]_2D_3$. Cell culture treatment with leptin resulted in no significant differences in osteoclast generation compared to the corresponding H7 group. Indomethacin showed no significant effect on TRAP (+) MNCs formation from the RAW264.7 cell line. PMA inhibited TRAP (+) MNCs formation induced by RANKL in the RAW264.7 cell culture. H7 had no effect on osteoclast generation from the RAW264.7 cell line. There was no difference compared with the corresponding control group after treatment with leptin. $1,25[OH]_2D_3$- or $PGE_2$-induced osteoclastic activity decreased significantly with leptin treatment at a concentration of 100 ng/ml in mouse bone marrow cell culture. Indomethacin, PMA, and H7 significantly inhibited osteoclastic activity induced by $1,25[OH]_2D_3$ in mouse bone marrow cell culture. No significant differences were found between the leptin treated group and the corresponding control group. The secretion of OPG, a substance known to inhibit osteoclast formation, was detected from the osteoblasts. Treatment by leptin resulted in significant increases in OPG secretion by osteoblastic cells. Taken these results, leptin may be an important regulatory cytokines within the bone marrow microenvironment.
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문제 정의
of OPG in osteoblastic cells. Additionally, this study examined the possible involvement between PGE2/ PKC-mediated signals and leptin on anti-osteoclastogenesis in various osteoclast culture systems.
in osteoblastic cells in vitro. This study examined the possible involvement between PGE2/PKC-mediated signals and leptin on anti-osteoclastogenesis. Osteoclast generation was measured by examining the number of TRAP (+) multinucleated cells (MNCs) after culturing osteoclast precursor cells on 96-well plates.
제안 방법
The present study was undertaken to determine the effects of leptin on anti- osteoclastogenesis using murine osteoclast precursors cultured on calcium-phosphate coated plates and on the production of OPG in osteoblastic cells in vitro. Additi onally, this study examined the possible involvement of PGE2/PKC-mediated signals on the effect of leptin on anti- osteoclastogenesis to various culture systems of osteoclast precursors.
The goal of the present study was to determine the effects of leptin on anti-osteoclastogenesis 니sing murine precursors cult니Ted on calcium-phosphate coated plates and on the regulation of OPG in osteoblastic cells. Additionally, this study examined the possible involvement between PGE2/ PKC-mediated signals and leptin on anti-osteoclastogenesis in various osteoclast culture systems.
To confirm that leptin could act indirectly on RANKL, we analyzed the effect of OPG. OPG is an osteoblast-secreted decoy receptor that inhibit osteoclast differentiation and activation.
대상 데이터
Recombinant human leptin and 1, 25- dihydroxycholecalciferol (1, 25[OH]2D3) were purchased from Biomol (PA, USA). Dexamethasone, Prostaglandin E2 (PGE2), indomethacin, phorbol 12-myristate 13-acetate (PMA), and isoquinoline-5-sulfdnic 2-methyl-l -piperazide dihydrochloride (H7) were purchased from Sigma-Aldrich Co. (MO, USA).
Six- to 8」week old male ICR mice were used. Media and fetal bovine serum (FBS) and other cultural reagents were purchased from Gibco laboratories (Grand Island, NY, USA).
Well-characterized osteoblastic cell lines, MG63 cells, were used in this study. MG63 cells were cultured in DMEM containing 10% FBS.
데이터처리
in each experiment. Data were analyzed using Student's t test. A P value less than 0.
성능/효과
In addition, leptin's dual local role of reducing resorption while increasing osteoblast differentiation (Gordeladze et al” 2002) suggest that it may have an important bone-conserving function. The results of this experiment suggest that a possible signaling pathway in which leptin plays a significant role, is in the regulation of the expression of OPQ but the precise molecular mechanisms associated with the anti-osteoclastogenesis effects of leptin have yet to be defined. The possibility needs to be explored further whether pharmacological manipulation of the leptin pathway may be a novel approach to treat osteoporosis.
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