두개골 및 두개봉합부 초기발육과정에서의 전사조절인자인 Msx2와 DIx5의 역할

송민호,박미현,남순현,김영진,류현모,김현정 大韓小兒齒科學會 2003 大韓小兒齒科學會誌 Vol.30 No.3

두개봉합부의 조기융합으로 일컬어지는 craniosynostosis는 두개봉합부에서의 골아세포의 조기분화 및 석회화의 결과로 나타나는 선천성 발육이상이다. 최근 유전학적 연구에 의하면 homeobox gene인 Msx2의 변이에 의해 Boston-type craniosynostosis가 야기되며, 또한 Dlx5 homozygote mutant mouse의 표현형에서 두개골의 골화지연을 포함한 다양한 두개안면부위의 이상을 발견하였다는 보고가 있었다. 게다가 Msx2와 Dlx5 homeodomain protein의 상호작용에 의해 성숙골아세포의 표지자인 osteocalcin의 전사를 조절할 수 있다는 사실이 알려져 있다. 이러한 일련의 결과들은 Msx2, Dlx5 및 osteocalcin 유전자들이 두개골의 골화과정과 두개봉합부의 형태발생에 중요한 역할을 담당하고 있음을 제시해주고 있다. 두개골의 성장과 두개봉합부의 형태발생시 이러한 유전자들의 기능을 알아보기위해 mouse의 태생기 (E15-E18) 동안 osteocalcin, Msx2, 및 Dlx5 유전자들의 발현양상을 조사하였다. Osteocalcin은 E15부터 두정골의 골막에서 관찰되었으며, 발생시기가 후기일수록 강한 발현양상을 나타내었다. Msx2는 시상봉합부의 미분화간엽조직과 osteogenic front에서 강하게 발현되었으며 경막과 hair follicle에서도 관찰되었다. Dlx5는 osteogenic front를 포함한 두정골의 골막에서 강하게 발현되었으나 시상봉합부의 미분화간엽조직에서는 발현되지않아, Msx2와는 발현양상의 차이를 나타내었다. 이 결과들을 종합해볼 때, Msx2와 Dlx5 유전자는 두개골과 두개봉합부의 성장발육과정에 중요한 역할을 담당하고 있으며, BMP signaling은 두 전사조절인자들을 조절하므로써 두개골의 골화과정과 두개봉합부의 형태발생 및 유지에 관여하고 있음을 제시해주고 있다. 특히 BMP signaling에 specific downstream gene인 Msx2 및 Dlx5의 발현양상의 차이는 골아세포의 분화시 이들 유전자가 각각의 독특한 기능을 가지고 있음을 시사해주고 있다. Craniosynostosis, known as a premature fusion of cranial sutures, is a developmental disorder characterized by precocious differentiation and mineralization of osteoblasts in the calvarial sutures. Recent genetic studies have demonstrated that mutation in the homeobox gene Msx2 causes Boston-type human craniosynostosis. Additionally, the phenotype of Dlx5 homozygote mutant mouse presents craniofacial abnormalities including a delayed ossification of calvarial bone. Furthemore transcription of osteocalcin, a mature osteoblast marker, is reciprocally regulated by the nomeodomain proteins Msx2 and Dlx5. These facts suggest important roles of osteocalcin, Msx2 and Dlx5 genes in the calvarial bone growth and suture morphogenesis. To elucidate the function of these molecules in the early morphogenesis of mouse cranial sutures, wer have first analyzed by in situ hybridization the expression of osteocalcin, Msx2 and Dlx5 genes in the developing parietal bone and sagittal suture of mouse calvaria during the embryonic (E15-E18) stage. Osteocalcin mRNA was found in the periosteum of parietal bones from E15, and gradually more highly expressed with aging. Msx2 mRNA was intensely expressed in the sutural mesenchyme, osteogenic fronts and midly expressed in the dura mater during the embryonic stage. Dlx5 mRNA was intensely expressed osteogenic fronts and the periostem of parietal bones. To further examine the upstream signaling molecules of transcription factor Msx2 and Dlx5, we have done in vitro experiments in E15.5 mouse calvarial explants. Interestingly, implantation of BMP2-, BMP4-soaked beads onto the osteogenic fronts after 48 hours organ culture induced etopic expressions of Msx2 and Dlx5 genes. On the other hand, overexpression of TGFβ1, GDF-6, -7, FGF-2, -4 and Shh did not induce the expression of Msx2 and Dlx5. Taken together, these data indicate that transcription factor Msx2 and Dlx5 play critical roles in the calvarial bone and suture development, and that BMP siganling is involved in the osteogenesis of calvarial bones and the maintenance of cranial sutures through these two transcriotpn factors. Furthermore, different expression patterns between Msx2 and Dlx5 suggest their specific functions in the osteoblast differentiation.

FGF-mediated FGFR signaling 이 두개봉합부의 초기형태발생 및 유지기전에 미치는 영향

남순현,김영진,서경환,김현정,박미현,유현모 大韓小兒齒科學會 1999 大韓小兒齒科學會誌 Vol.26 No.4

두개봉합부의 조기융합으로 알려진 Craniosynostosis는 두개봉합부 주위 조직들 사이의 조화로운 상호 작용이 파괴되었을 때 야기될 수 있다. 흥미롭게도 FGF receptor들, 특히 FGFR2의 point mutation은 여러 가지 형태의 craniosynostosis 증후군과 연관되어 있어, FGFR가 두개봉합부를 포함한 두개골 성장 발달과정에 중요한 유전자임을 시사하고 있다. Mouse 두개봉합부의 초기형태발생시 FGFR 유전자들의 기능을 알아보기위해, in situ hybridization 방법을 이용하여 FGFR2(BEK) 및 골아세포분화의 초기표지자인 osteopontin이, 태생기(E15-18)에서 출생후(P1-P3)까지, 두개골의 시상봉합부에서의 발현양상을 조사하였다. FGFR2(BEK)은 osteogenic front에 강하게 발현되었으며, osteopontin은 parietal bone의 exo-, endocranial부위에서 발현되었으나, parietal bone의 성장가장자리인 osteogenic front에서는 관찰되지 않았다. 두개봉합부에서의 FGF-mediated FGFR signaling의 역할을 좀더 심도깊게 조사 하기위해 E15.5 mouse의 두개골을 이용하여 in vitro 실험을 시행하였다. 흥미롭게도 osteogenic fronts 및 시상봉합부의 간엽조직 중앙에 FGF2 - soaked beads를 점적하여 36시간 기관배양한 결과, bead주위 조직들의 두께 및 세포수가 증가되었으며, osteogenic fronts 상에 FGF4 beads를 올려놓은 경우, 시상두개봉합부 중앙에 점적된 FGF4 beads나 BSA control beads에 비해, 골성장이 촉진되어 시상두개봉합부의 부분적인 소멸을 관찰할 수 있었다. 이와 더불어 FGFR2 beads는 osteopotin 및 Msxl 유전자의 발현을 유도하였다. 이 결과들을 종합해 볼 때, FGF - mediated FGFR signaling이 발육중인 두개골과 두개봉합부에서 세포의 증식과 분화의 균형을 조절하는데 중요한 담당하고 있음을 시사해주고 있으며, 이 과정중 FGF signaling이 osteopontin 및 Msxl 유전자의 발현을 조절하므로써 막내골 성장 및 두개봉합부의 유지기전에 기여할 것으로 사료된다. Craniosynostosis, the premature fusion of cranial sutures, presumably involves disturbance of the interactions between different tissues within the cranial sutures. Interestingly, point mutaions in the genes encoding for the fibroblast growth factor receptors(FGFRs), especially FGFR2, cause various types of human craniosynostosis syndromes. To elucidate the function of these genes in the early morphogenesis of mouse cranial sutures, we first analyzed by in situ hybridization the expression of FGFR2(BEX) and osteopontin, an early marker of osteogenic differentiation, in the sagittal suture of calvaria during embryonic (E15-E18) and postnatal stage (P1 - P3). FGFR2(BEK) was intensely expressed in the osteogenic fronts, whose cells undergo differentiation into osteoprogenitor cells that ultimately lay down the bone matrix. Osteopontin was expressed throughout the parietal bones excluding the osteogenic fronts, the periphery of the parietal bones. To further examine the role of FGF-mediated FGFR signaling in cranial suture, we did in vitro experiments in E15.5 mouse calvarial explants. Interestingly, implantation of FGF2 soaked beads onto both the osteogenic fronts and mid-mesenchyme of sagittal suture after 36 hours organ culture resulted in the increase of the tissue thickness and cell number around FGF2 beads, moreover FGF4-soaked beads implanted onto the osteogenic fronts stimulated suture closure due to an accelerated bone growth, compared to FGF4 beads placed onto mid-mesenchyme of sagittal suture and BSA control beads. In addition FGF2 induced the ectopic expression of osteopontin and Msxl genes. Taken together, these data indicate that FGF-mediated FGFR signaling has a important role in regulating the cranial bone growth and maintenance of cranial suture, and suggest that FGF-mediated FGFR signaling is involved in regulating the balance between the cell proliferation and differentiation through inducing the expression of osteopontin and Msxl genes.

FGF signaling이 연골 형성에 미치는 영향

박충제,이상원,남순현,김영진,류현모,김현정 大韓小兒齒科學會 2003 大韓小兒齒科學會誌 Vol.30 No.4

막내골화와 연골내골화 등의 정상적인 골격 성장은 섬유아세포 성장인자 (FGF) 와 이들 수용체들 (FGFR) 에 의한 신호전달체계에 의해 조절된다. 또한 전사조절인자인 Runx2 (Cbfa1/Pebp2αA/AML3) 는 골아세포분화 뿐만 아니라 골형성에도 필수적인 유전자로 알려져 있다. FGF signaling이 mouse의 두 개관과 하악에서의 연골 형성에 어떤 영향을 미치고 있으며, 이 과정에서 Runx2의 연관성을 알아보고자 in vivo 및 in vitro 실험을 시행하여 다음과 같은 결과를 얻었다. 두 개관의 하악을 Alcian blue 염색한 결과 시상두개봉합부 연골은 태생16일부터, Meckel's 연골은 태생11일부터 형성되기 시작하였다. 이들 연골세포들의 성사을 알아보기위한 in situ hybridization 결과 시상두개봉합부 연골 및 Meckel's 연골 모두에서 type Ⅱ collagen은 발현되었으나, Type X collagen은 발현되지 않았다. Runx2 mRNA는 시상두개봉합부 연골과 Meckel's 연골 모두에서 발현되지 않았지만, 이들 연골들의 가장자리를 둘러싸고 있는 독특한 발현양상을 나타내었다. 두개 봉합부에서의 FGF2 protein의 국소적 적용은 두개봉합부 하방의 연골형성을 억제하였다. 또한 하악의 Meckel's 연골 발생부위에 FGF2 protein의 국소적 적용 역시 Meckel's 연골의 형성을 억제하였다. FGF2 protein은 시상두개봉합부상의 bead 주위로 Runx2의 발현을 유도하였다. 이 결과들을 종합해볼 때, FGF signaling은 골아세포와 연골아세포가 공존하고 있는 조직에서의 연골 형성을 억제하고 있음을 시사해 주고 있으며, 이 과정에서 FGF signaling은 부분적으로 Runx2 유전자의 발현을 조절하므로써 연골세포의 증식과 분화에 관여할 것으로 사료된다. Figroblast growth factor (FGF) / FGF receptor (FGFR) mediated signaling is required for skeletogenesis including intramembranous and endochondral ossifications. Runx2 (Cbfa1/Pebp2αA/AML3) is an essential transcription factor for osteoblast differentiation and bone formation. Murine calvaria and mandible are concurrently undergoing both intramembranous bone and cartilage formations in the early development stage. However the mechanism by which these cartilage formations are regulated remains unclear. To elucidate the effect of FGF signaling on development of cranial sutural catilage and Meckel's cartilage and to understand the role of Runx2 in these processs, we have done both in vivo and in vitro experiments. Alcian blue staining showed that cartilage formation in sagittal suture beings from embryonic state 16 (E16), Meckel's cartilage formation in mandible from E12. We analyzed by in situ hybridization the characteristics of cartilage cells that type Ⅱ collagen, not type X collagen, was expressed in sagittal sutural cartilage and Meckel's cartilage. In addition, Runx2 was not expressed in Meckel's cartialge as well as sagittal sutural cartilage, except specific expression pattern only surrounding both cartilages. FGF signaling pathway was further examined in vitro. Beads soaked in FGF2 placed on the sagittal suture and mandible inhibited both sutural and Meckel's cartilage formations. We next examined whether Runx2 gene lies in FGF siganling pathway during regulation of catilage formation. These results suggest that FGF signaling inhibits formations of sagittal sutural and Meckel's cartilages, also propose that FGF siganling is involved in the proliferation and differentiation of chondroblasts through regulating the transcription factor Runx2.

광원의 종류에 따른 복합레진의 중합거동 및 중합률에 관한 연구

류주희,이인복,유현미,김미자,석창인,권혁춘 大韓齒科保存學會 2004 Restorative Dentistry & Endodontics Vol.29 No.4

Objectives: The purpose of this study was to observe the reaction kinetics and the degree of polymerization of composite resins when cured by different light sources and to evaluate the effectiveness of the blue Light Emitting Diode Light Curing Units (LED LCUs) compared with conventional halogen LGUs. Materials and Methods: First, thermal analysis was performed by a differential scanning calorimeter(DSC). The LED LCU (Elipar Freelight, 320㎽/㎠) and the conventional halogen LCU (XL3000, 400㎽/㎠) were used in this study for curing three composite resins (SureFil, Z-250 and AEliteFLO). Second, the degree of conversion was obtained in the composite resins cured according to the above curing mode with a FTIR. Third, the measurements of depth of cure were carried out in accordance with ISO 4049 standards. Statistical analysis was performed by two-way ANOVA test at 95% levels of confidence and Duncan's procedure for multiple comparisons. Results: The heat of cure was not statistically different among the LCUs (p > 0.05). The composites cured by the LED (Exp) LCUs were statistically more slowly polymerized than by the halogen LCU and the LED (Std) LCU (p< 0.05). The composite resin groups cured by the LED (Exp) LGUs had significantly greater degree of conversion value than by the halogen LCU and the LED (Std) LCU (p = 0.0002). The composite resin groups cured by the LED (Std) LGUs showed significantly greater depth of cure value than by the halogen LCU and the LED (Exp) LGU (p < 0.05).

Sosteoblast 분화에 미치는 BMP-2의 역할

류현모 대한내분비학회 2001 Endocrinology and metabolism Vol.16 No.4-5

태권도 돌려차기 동작시 숙련자와 비숙련자간 단계별 주동근의 동원양상 및 피로도 분석

류병관,박현식 龍仁大學校 武道硏究所 2003 武道硏究所誌 Vol.14 No.1

The purpose of this study was to verify what major muscles are during Taekwondo dolyechagi, and analyze major muscle fatigue and type on recruitment. 14 university students(male) were participated in the exercise test. The subject were divided into two groups; expert (N=7) and non-expert(N=7). Electromyogram test was analyzed muscle fatigue and type on recruitment during repeated dolyechagi by surface electrode. Major muscle was measured by Integrated EMG in main-test while dolyechagi 3 times. There were analyzed by Integrated EMG on Major muscle when lst, 2nd, and 3rd time of 100. Muscle fatigue was analyzed by MPF(mean power frequency) on Major muscle when dolyechagi lst, 20th, 40th, 60th, 80th, 90th and 100th time of 100. The results of this study as follows: 1. When Taekwondo dolyechagi, this study showed that major muscle of left foot was measured Left Tibialis anterior, Left Rectusfemoris, Left biceps femoris, Left gastrocnemius and major muscle of Right foot was Right Tibialis anterior, Right Rectusfemoris, Right gastrocnemius and Right biceps femoris. 2. When Taekwondo dolyechagi, this study showed that major muscle of left foot and Right foot was measured Tibialis anterior, Rectusfemoris. 3. non-expert group in dolyechagi significantly reunited more major muscles of Right foot than in expert group. However, expert group in dolyechagi significantly recruited more Left Tibialis anterior, Left Rectusfemoris than in non-expert group. 4. The change of the MPF in major muscles of LeftㆍRight Tibialis anterior, Leftㆍ Right Rectusfemoris were more statistically significant in dolyechagi than kuzu shi. This results suggested that muscle fatigue occured more in kake. The difference of MPF between non-expert group and expert group was resulted from high resistant capacity in expert group.

可姙期 女性의 唾液中 Peroxidase, N-acetyl-β-D-glucosaminidase의 活性度 및 遊離 sialic acid 含量의 週期的 變動

柳顯模,卞鍾秀,曺準承 慶北大學校 齒科大學 1990 慶北齒大論文集 Vol.7 No.1

In order to determine the possible effects of hormonal changes on the salivary components, the activities of peroxidase and N-acetyl-β-D-glucosaminidase, and the content of free sialic acid were measured in saliva during menstrual cycles of 11 healthy women. Elevations of salivary peroxidase and N-acetyl-β-D-glucosaminidase activities were found around the time of ovulation in 8 cycling woment : the highest enzymatic activities which were measured 15.38, 14.63 days before menstruation were 118.76±10.01 Unit/min/g of protein, 21.95±1.04 nmol/min/mg of protein in peroxidase and N-acetyl-β-D-glucosaminidase, respectively. These value of highest activity significantly increased, compared to those of menstrual period, 38.24% and 21.48% in peroxidase and N-acetyl-β-D-glucosaminidase, resectively. Large cyclic variation without any clear tendency occured in the content of the free sialic acid. J. Kyungpook Univ. Sch. Dent. Vol. 7, No. 1,23∼32, 1990

Rat에 있어서 사료내 아연의 첨가가 조직내 카드뮴의 농도 및 혈액상에 미치는 영향

류정열,이현범,이근우 경북대학교 1990 새마을 硏究論叢 Vol.11 No.-

Present experiment was undrtaken in order to clarify the effect of supplemental zinc on the absorption of cadmium in diet and hematological picture. The rats were fed with diet containing 10㎍/g of cadmium(Control rats) and the other 10 with the same diet which were added with 2,000㎍/g of zinc(Experimental rats). One half of the rats were sacrified on 30th day and the remainder on 60th day. Kidney, liver, muscle, and hair samples were collected, ashed and analyzed for the cadmium and zinc contents using an atomic absorption spectrophotometer. In addition, blood samples collected at necropsy were examined for erythrocyte count, complete leukocyte count and packed cell volume. The result obtained are summrized as follows: The mean cadmium contents of control group were measured as 0.91㎍/g in liver, 1.23㎍/g in kidney, 0.35㎍/g in muscle and 2.1㎍/g in hair. The mean cadmium contents of experimental group were measured as 0.86㎍/g in liver, 1.49㎍/g in kidney, 0.11㎍/g in muscle and 0.16㎍/g in hair. These values showed no significant difference statistically between the two groups. The mean zinc contents of control group were measured as 11.39㎍/g in liver, 26.82㎍/g in kidney, 13.14㎍/g in muscle and 60.27㎍/g in hair. The mean zinc contents of experimental group were measured as 12.75㎍/g in liver, 22.06㎍/g in kidney, 10.17㎍/g in muscle and 70.92㎍/g in hair. No significant difference was recorgnized between the two groups. The mean hematological pictures of control group were measured as 764×10 exp (4)/cmm in erythrocyte count, 13,830cmm in leukocyte count, 40% in packed cell volume, 12% in neutrophill count, 88% in lymphocyte count and 1% in monocyte count. The mean hematological pictures of experimental group were measured as 686×10 exp (4)/cmm in erythrocyte count, 10,905cmm in leukocyte count, 40% in packed cell volume, 17% in neutrophil count, 83% in lymphocyte count, and 1% in monocyte count. A significant difference(p<0.1) was observable only in the erythrocyte counts of the two groups.

파골세포 활성에 있어 gp130과 PTH의 연관성

김영준,류현모,김신윤,신홍인 경북대학교 병원 2002 경북대학교병원의학연구소논문집 Vol.6 No.1

gp130,a subunit for serveral cytokine signal transducing receptors, mediates diverse activities including osteoclastogenesis. In vitro obaervations suggest that PTH also triggers the gp130-mediated signal for bone resorption by stimulating secretion of IL-6,IL-11 and LIF from osteoblast/stromal cells. Though this gp130 signaling is required for full activation of bone resporption by PTH in vitro, the relationship between PTH and gp130 signaling pathway in vitro is not yet clanified. To address the importance of gp130 as a mediator of PTH action in osteoclast activation, the blood ionized calium and blood serum PTH level was investigated in gp130-/- and wild type mice at E18.5 using a CIBA/Coming 634 Ca^+2/pH analyzer and rat intact PTH Kit, respectively.In addition, calium transport efficiency was assessed from gp130 mice at E17.5. The resorptive activity of osteoclasts from intact calvariae by a ^45Ca release assay and the osteoclastic developmental condition in tibiae were also analyzed, respectively.The gp130-/- micerevealed significantly lower ionized calcium level(1.44±0.03 vs1.77±0.03 mmol/ℓ, p<0.001)and significantly higher blood serum PTH(251.8±5.3 VS 20.5±5.3 pg/㎖,p<0.001) compared to wild type littermates.There was no remarkable pathologic change of placenta in gp130-/- and the ^45Ca transport though placenta was accelerated in gp130-/- compared to wild type littermates(%to Het 83%in -/- vs 148% in +/+).The gp130-/- embryos had small skeletons with bones containing little primary spongiosa, and large multinucleated TRAP+ cells along the lower border of the growth plate. Release of ^45Ca from prelableled calvarial bone(E18.5)in response to either 10^-7 M hpTH(1-34)or 20 ng/㎖ sRANKL was lower in gp130-/-calvarial bone compared to gp130+/+ calvarial bone (PTH 1.83±0.6 fold over basal in-/- vs 2.79±0.6 in +/+and sRANKL1.3±0.4 in -/- vs 2.79±0.6 in +/+ and sRANKL1.3±0.4 in -/- vs 2.0±0.4 in +/+,p<0.5). These result strongly support the hypothesis that PTH activates bone resortion,in part, by stimulating ostedtrophic cytokine secretiom from osteoblast/stromal cells, which tirggers gp130-mediated signaling for osteoclastic activation.

Smad에 의한 alkaline phosphatase 유전자의 발현 조절기전

김난진,류현모,김현정,김영진,남순현 대한소아치과학회 2001 大韓小兒齒科學會誌 Vol.28 No.2

본 실험은 탁월한 골유도능으로 관심의 대상이 되고 있는 RMP의 세포내 신호 전달자로 알려진 Smad 1과 Smad 5가 조 골세포 초기 분화표지인자인 ALP 유전자의 발현에 미치는 영향 및 그 조절기전을 알아보고자 하였다. BMP 처리 없이도 Smad에 의해 ALP가 발현되는가를 알아보기 위해 Smad 1과 Smad 5가 각각 stably transfection된 C2C12 세포를 3일간 배양후 histochemical assay를 하였고, Smad 1과 Smad 5의 expression vector와 ALP promoter vector를 transient co-transfectiongksgn ALP promoter activity를 측정하였다. Smad에 의한 BMP의 효과를 알아보기 위해서 100ng/ml의 BMP-2를 처리한 군과 처리하지 않은 군으로 나누어 세포를 배양한후 ALP 유전자의 발현을 northern blot analysis로 확인하였다. Smad가 ALP 유전자의 발현을 직접적으로 조절하는가를 알아보기 위여서는 단백질 합성억제제인 cycloheximide를 전처리하여 ALP 유전자의 발현을 northern blot analysis하였다. 이상의 실험결과 다음과 같은 결론을 얻었다. ·Smad 1과 Smad 5가 과발현된 세포에서는 BMP 처리없이도 ALP가 발현된다. ·Smad 1과 Smad 5가 과발현된 세포에서 RMP 처리후 ALP 발현 증가율이 대조군 보다 현저히 높게 나타나 Smad가 BMP 효과를 증가시킨다는 것을 알수있다. ·Smad는 새로운 단백질의 합성을 통해 ALP 유전자를 발현시킨다. Bone morphogenetic proteins(BMPs), members of the transforming growth factor β(TGF-β) superfamily were first identified as the factors that induce ectopic bone formation in vivo, when implanted into muscular tissue. Especially BMP-2 inhibits terminal differentiation of C2C12 myoblasts and converts them into osteoblast lineage cells. In the molecular mechanism of the signal transduction of TGF-β and related factors, intracellular signaling proteins were identified as Smad. In previous study, it has been reported that Smad 1 and Smad 5, which belong to the R-Smad family mediate BMP signaling, were involved in the induction of osteoblast differentiation in C2C12 cells. To understnad the role of Smads involved in osteogenic transdifferentiation in C2C12 cell, in present study, after we stably transfected C2C12 cells with each. Smad(Smad 1, Smad 5) expression vector, cultured for 3 days and stained for alkaline phophatase activity. ALP activity positive cells appeared in the Smad 1, Smad 5 stably transfected cell even in the abscence of BMP. After transiently co-transfected C2C12 cells with each Smad expression vector and ALP promoter, it was excence of MBP. These result suggested that both Smad 1 and Smad 5 were involved in the intracellular BMP signals which induce osteoblast differentiation in C2C12 cells. The effect of BMP on C2C12 cells with Smad 1, Smad 5 transfected were studied by using northern blot analysis. the treatment of BMP upregulated ALP mRNA level in three groups, especially upregulation of ALP was larger in Smad 1, Smad 5 transfected cell than control group. Pretreatment with cycloheximide(10㎍/ml), a protein synthesis inhibitor resulted in blocking the ALP gene expression even in BMP(100ng/ml) treated cell. These results suggested that Smad increased the level of ALP mRNA via the synthesis of a certain transcriptional regulatory protein.