Chondrogenesis of human umbilical cord-derived mesenchymal stem cells in vitro by TGFβ3 and BMP6

Eu-Gene Park,조태준,Soon-Keun Kwon,Dong-Sup Lee,조재진 충북대학교 동물의학연구소 2012 Journal of Biomedical and Translational Research Vol.13 No.4

Human umbilical cord is easy to obtain because it is discarded after birth, so it can avoid the ethical issues. Chondrogenesis studies using MSCs from bone marrow, cord blood, adipose has been indicated that TGFβ3 and BMP6 stimulate chondrogenesis. Therefore, we investigated chondrogenesis of the hUC-MSCs on TGFβ3, BMP6 and combination of the two growth factors. We initiated chondrogenesis of the cells by giving physical forces to form 3D cell clusters. After the initiation, we gave four experimental groups for differentiation of the cells as follows; control, 10 ng/mL TGFβ3, 100 ng/mL BMP6, and the combination of 5 ng/mL TGFβ3 and 50 ng/mL BMP6. To analyze of the chondrogenesis, GAG contents, mRNA expression, histological and immunohistochemistry (IHC) were performed. To analyze GAG contents, GAG assay was measured and RT-PCR was performed to determine the chondrogenic markers. Histological analysis was performed through safranin O, alcian blue, and IHC was performed using collagen type I and II. GAG contents were increased that 184% by TGFβ3, 147% by BMP6 and 189% by the combination of TGFβ3 and BMP6 compared to control. The growth factors improved collagen II and aggrecan expression, especially, TGFβ3 and BMP6 were shown synergistic effect compared to only TGFβ3 or BMP6 treated. The histological and IHC analysis indicated that the chondrogenic differentiation in the TGFβ3 and the combination of TGFβ3 and BMP6 showed more cartilage deposition. In conclusion, TGFβ3 and BMP6 differentiated hUC-MSCs into chondrogenic clusters of the combination treatment of the two growth factors showed more efficient chondrogenic ability.

Analysis and characterization of the functional TGFβ receptors required for BMP6-induced osteogenic differentiation of mesenchymal progenitor cells

( Yan Zhang ),( De Ying Zhang ),( Yan Fang Zhao ),( Jin Wang ),( Juan Wen He ),( Jin Yong Luo ) 생화학분자생물학회 2013 BMB Reports Vol.46 No.2

Although BMP6 is highly capable of inducing osteogenic differentiation of mesenchymal progenitor cells (MPCs), the molecular mechanism involved remains to be fully elucidated. Using dominant negative (dn) mutant form of type I and type II TGFβ receptors, we demonstrated that three dn-type I receptors (dnALK2, dnALK3, dnALK6), and three dn-type II receptors (dnBMPRII, dnActRII, dnActRIIB), effectively diminished BMP6- induced osteogenic differentiation of MPCs. These findings suggested that ALK2, ALK3, ALK6, BMPRII, ActRII and ActRIIB are essential for BMP6-induced osteogenic differentiation of MPCs. However, MPCs in this study do not express ActRIIB. Moreover, RNA interference of ALK2, ALK3, ALK6, BMPRII and ActRII inhibited BMP6-induced osteogenic differentiation in MPCs. Our results strongly suggested that BMP6-induced osteogenic differentiation of MPCs is mediated by its functional TGFβ receptors including ALK2, ALK3, ALK6, BMPRII, and ActRII. [BMB Reports 2013; 46(2): 107-112].

The Identification of a Receptor-binding Peptide Derived from Bone Morphogenetic Protein-6 and Its Role in Osteogenesis

최윤정,이주연,Chong Pyoung Chung,박윤정 한국생체재료학회 2014 생체재료학회지 Vol.18 No.1

Bone morphogenetic protein (BMP)-6 induces the differentiation of mesenchymal-derived cells into osteochondrogenic cells. Similar to the potent osteochondrogenic growth factor BMP-2, the BMP-6 also contains the “knuckle epitope”, which is thought to bind BMP receptor type II. In this study, we analyzed a synthetic peptide, YVPLPCCAPTKLNAISVLYF, namely BMP receptor binding peptide (BRBP), corresponding to residues of XX-YZ of the knuckle epitope of BMP-6. Our data revealed that the BRBP significantly binds to both type I and type II BMP receptors(BMPRs) with a similar affinity to BMP-6. The BRBP also induces osteogenic differentiation of human mesenchymal stem cells (HMSCs) with similar potency to BMP-6 as measured by alkaline phosphatase (ALP) activity, mineralization and osteogenic gene marker expression. Furthermore, the BRBP stimulates the phosphorylation of both the extracellular signal-regulated kinase (ERK1/2), and the Smad1/5/8 proteins. Blocking ERK-signaling with U0126, an inhibitor for MAPK/ERK kinase (MEK), abolishes the oseteogenic effects of the BRBP on human mesenchymal stem cells (HMSCs). Thus our study identifies an osteogenic peptide derived from BMP-6, which may be useful for ossified tissue regeneration.

Expression of BMP6 is Associated with its Methylation Status in Colorectal Cancer Tissue but Lacks Prognostic Significance

Sangplod, Patcharaporn,Kanngurn, Samornmas,Boonpipattanapong, Teeranut,Ruangrat, Pritsana,Sangkhathat, Surasak Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.17

Background: The study aimed to evaluate the incidence of CpG island promoter methylation of BMP6, a member of the transforming growth factor beta family, in tissue samples from colorectal cancers (CRC) and look for its association with BMP6 expression and clinicopathological correlation. Materials and Methods: Methylation specific PCR for the BMP6 promoter region was performed with 85 frozen tissue samples of CRC and 45 of normal colon. Methylation status of MLH1 was also determined by the same method. Expression of BMP6 was evaluated by immunohistochemistry (IHC), using Allred's scoring system. The methylation status was analyzed against clinical and pathological parameters in CRC. Results: The study revealed BMP6 hypermethylation in 34 of 85 tumor specimens (40%), and 15 out of 45 normal tissue samples from CRC (33%). The incidence of hypermethylation was inversely correlated with IHC score. Allred's scores of 7 or more were correlated with lower frequency of BMP6 hypermethylation (29% compared to 50% in the remaining, p-value 0.049). However, there was no association between hypermethylation status and any clinicopathological parameters. The methylation status of BMP6 was not correlated with that of MLH1, a key methylation determinant in CRC. On survival analysis, there was no significant difference in progress-free survival (PFS) between the cases with and without hypermethylation (2-year PFS 74% and 76%, respectively). Conclusions: CpG island methylation of BMP6 is found in high frequency in CRC and this epigenetic event is associated with suppressed protein expression in the tumor tissue. However, the marker is not associated with tumor progression of the disease.

Hoxc8 Represses BMP-Induced Expression of Smad6

Kang, Myeng-Mo,Bok, Jin-Woong,Deocaris, Custer C.,Park, Hyoung-Woo,Kim, Myoung-Hee Korean Society for Molecular and Cellular Biology 2010 Molecules and cells Vol.29 No.1

Proper regulation of bone morphogenetic protein (BMP) signaling is critical for correct patterning and morphogenesis of various tissues and organs. A well known feedback mechanism is a BMP-mediated induction of Smad6, an inhibitor of BMP signaling. Hoxc8, one of the Hox family transcription factors, has also been shown to negatively regulate BMP-mediated gene expression. Here we add another level of Hoxc8 regulation on BMP signaling. Our results show that Hoxc8, when over-expressed in C3H10T1/2 or C2C12 cells, suppressed basal Smad6 promoter activity and its mRNA expression. Activation of Smad6 transcription either by BMP2 treatment or Smad1 over-expression was also abolished by Hoxc8. When chromatin was precipitated from mouse embryos with anti-Smad1 or anti-Hoxc8 antibody, Smad6 promoter sequence was enriched, suggesting that Hoxc8 proteins make complexes with Smad1 in the Smad6 promoter region. Yet, a lack of Hox binding motifs in the Smad6 promoter sequence suggests that instead of directly binding to the DNA, Hoxc8 may regulate Smad6 expression via an indirect mechanism. Our results suggest that the Smad6-mediated negative feedback mechanism on BMP signaling may be balanced by the repression of Smad6 expression by Hoxc8.

가토 상악동 점막 거상 후 DBBP를 이식재로 사용시 BMP4, BMP6의 발현

이현석,허현아,표성운,이원,Lee, Hyun-Suk,Heo, Hyun-A,Pyo, Sung-Woon,Lee, Won 대한악안면성형재건외과학회 2007 Maxillofacial Plastic Reconstructive Surgery Vol.29 No.6

이 실험은 이식재로 Deproteinated Bovine Bone Powder (DBBP)를 사용하였을 때와 골 이식재를 사용하지 않고 단순히 Absorbable Gelatin Sponge(AGS)만을 사용하였을 때의 BMP4와 BMP6의 상대적인 발현량을 real-time RT-PCR을 이용하여 비교해 보기 위한 것으로 다음과 같은 결과를 얻었다. 1. BMP4의 경우 처음 1일째와 3일째의 경우 DBBP군과 AGS군 모두 일정하게 증가하였으나 5일째 AGS군에서 감소하다가 7일째 다시 증가하였으며 9일째 다시 감소하였다. DBBP군에서는 7일까지 계속하여 증가하다가 9일째 감소하였다. DBBP군이 AGS군에 비해 발현의 양이 많은 경우가 많았지만 유의성은 없었다 (p>0.05). 2 BMP6의 경우 처음 1일째와 3일째의 경우 DBBP군과 AGS군 모두 일정하게 증가하였으나 5일째 AGS군에서 감소하다가 7일째 다시 증가하였으며 9일째 다시 감소하였다. DBBP군에서는 7일까지 계속하여 증가하다가 9일째 감소하였다. AGS군이 DBBP군에 비해 발현의 양이 많은 경우가 많았지만 유의성은 없었다 (p>0.05). 3. 두 군간에 동일시기에 BMP발현이 유의할 만한 차이를 보이지 않는 것은 DBBP와 AGS 모두 space retainer로서 작용을 하여 혈병중의 BMP발현의 양상이 비슷하기 때문으로 여겨진다. 4. 따라서 DBBP가 AGS에 비해 초기 골재생에 크게 유리한 점은 없는 것으로 여겨지며 초기 골 형성에서 BMP의 발현은 이식재의 종류가 아니라 물리적인 carrier로서의 작용이 더 중요한 것으로 여겨진다. The most important factor for successful implantation is osseointegration between the implant and bone. The expression of bone morphogenetic proteins (BMPs) inducing bone formation would differ after maxillary sinus elevation. And within the same graft material. the expression of BMPs would change with time after graft. The aim of this study was to compare the relative expressions of BMP4 and BMP6 using real-time RT-PCR when maxillary sinus elevation was performed using deproteinated bovine bone powder (DBBP) as the graft material or absorbable gelatin sponge (AGS) as the filler without any graft material. Fifteen rabbits, each weighing between 3.0 to 3.5 Kg, were divided randomly into 5 groups of 3 animals each based on their time of sacrifice 0, 3, 5, 7 and 9 days). After exposure of the maxillary sinus bilaterally, bone graft was performed in the right maxillary sinus using DBBP ($BBP^{(R)}$ Oct Inc., Cheonan, Korea) and only AGS ($Gelfoam^{(R)}$ Pharmacia & Upjohn Company, Kalamazoo, MI, U.S.A) was placed into the left without any graft material. Each group of rabbits was sacrificed at 1, 3, 5, 7, or 9 days after operation and all specimens were harvested. And the following results were obtained using real-time RT-PCR from isolated total RNA of the samples. 1. The expression of BMP4 increased at postoperative 1 and 3 days in both DBBP group and AGS group. In AGS group. it decreased at postoperative 5 days. increased again at postoperative 7 days, and decreased at postoperative 9 days. In DBBP group, it increased until postoperative 7 days and decreased at postoperative 9 days. Although the expression of BMP4 was higher in DBBP group compared with AGS group, it was not statistically significant (p>0.05). 2. The expression of BMP6 increased at postoperative 1 and 3 days in both DBBP group and AGS group. In AGS group, it decreased at postoperative 5 days, increased again at postoperative 7 days, and decreased at postoperative 9 days. In DBBP group, it increased until postoperative 7 days and decreased at postoperative 9 days. Although the expression of BMP6 was higher in AGS group compared with DBBP group, it was not statistically significant (p>0.05). 3. There was no statistically significant difference in BMP expression in both groups during same period of time. It' s probably because DBBP and AGS both functioned as a space retainer so that the BMP expression in blood clot seemed to be similar. 4. Thus, DBBP would not offer many benefits for early bone regeneration compared with AGS. The expression of BMP in early bone formation seems to be more influenced by physical carrier rather than the graft type.

Hoxc8 Represses BMP-Induced Expression of Smad6

Myengmo Kang,복진웅,Custer C. Deocaris,박형우,김명희 한국분자세포생물학회 2010 Molecules and cells Vol.29 No.1

Proper regulation of bone morphogenetic protein (BMP) signaling is critical for correct patterning and morphogene-sis of various tissues and organs. A well known feedback mechanism is a BMP-mediated induction of Smad6, an in-hibitor of BMP signaling. Hoxc8, one of the Hox family tran-scription factors, has also been shown to negatively regu-late BMP-mediated gene expression. Here we add another level of Hoxc8 regulation on BMP signaling. Our results show that Hoxc8, when over-expressed in C3H10T1/2 or C2C12 cells, suppressed basal Smad6 promoter activity and its mRNA expression. Activation of Smad6 transcription either by BMP2 treatment or Smad1 over-expression was also abolished by Hoxc8. When chromatin was precipitated from mouse embryos with anti-Smad1 or anti-Hoxc8 anti-body, Smad6 promoter sequence was enriched, suggesting that Hoxc8 proteins make complexes with Smad1 in the Smad6 promoter region. Yet, a lack of Hox binding motifs in the Smad6 promoter sequence suggests that instead of directly binding to the DNA, Hoxc8 may regulate Smad6 expression via an indirect mechanism. Our results suggest that the Smad6-mediated negative feedback mechanism on BMP signaling may be balanced by the repression of Smad6 expression by Hoxc8.

BMP 신호 하부 인자인 PV.1 단백질의 결합 단백질 동정

황유석(Yoo-Seok Hwang),채정필(Jeong-Pil Chae),김동선(Dong-Sun Kim),박권무(Kwon Moo Park),배용철(Yong Chul Bae),박매자(Mae Ja Park) 대한해부학회 2007 Anatomy & Cell Biology Vol.40 No.3

척추동물에서 BMP의 배(ventral)쪽 신호 전달 경로의 하위에 있는 전사조절인자들은 등배(dorsalventral)쪽 축을 따라 형성되는 중배엽에 있어서도 그 기능을 공유 한다는 것이 보고 되었다. 본 연구에서는 BMP 신호 물질의 하위 유전자인 PV.1 단백질이 배쪽과 등쪽에서의 신호전달에 있어서 차별적 기능을 알기 위하여 PV.1과 상호 결합하는 단백질을 검색하였다. 효모 이중 접합법을 이용하여 24개의 PV.1 결합 단백질을 검색하였고, 이중 결합하는 Xvent-2와 Xclaudin-6에 대한 PV.1 단백질의 도메인(domain) 연구를 하였다. PV.1 단백질의 C-terminus인 197-241 지역이 Xclaudin-6와 결합하는 반면에 Xvent-2는 PV.1 단백질의 C-terminus 전반에서 결합한다는 것을 확인하였다. 또한 PV.1과 결합하는 Xvent-2는 동형 이중 결합(Homodimerization)뿐만 아니라 PV.1과 결합하는 Xclaudin-6과도 이형 이중 결합(Heteodimerization)을 한다는 것을 확인하였다. Homeodomain transcription factors functioning downstream of BMP ventral pathway have been reported to share similar domain of roles in mesoderm patterning along the dorsal-ventral axis. To elucidate the differential role of PV.1 in the aspect of relationship between dorsal and ventral region, we tried to screen PV.1- interacting proteins. Twenty-four PV.1-interacting proteins were identified by yeast two-hybrid screening. Xvent-2 and Xclaudin-6 among these, went under domain study. The C-terminus of PV.1, more specifically 197-241 region was found to interact with Xclaudin-6. Meanwhile Xvent-2 has mild affinity to overall C-terminal region of PV.1. At the same time it was found that Xvent-2 homodimerizes and also binds to Xclaudin-6.

Bone morphogenetic protein 6-induced interleukin-1β expression in macrophages requires PU.1/Smad1 interaction

Lee, G.T.,Jung, Y.S.,Lee, J.H.,Kim, W.J.,Kim, I.Y. Pergamon Press 2011 Molecular immunology Vol.48 No.12

Interleukin 1β (IL-1β) is a pro-inflammatory cytokine secreted by activated macrophages and monocytes. Previously, we have reported that bone morphogenetic protein-6 (BMP-6) induces inducible nitric oxide synthase (iNOS) expression via IL-1β in macrophages. In the present study, we demonstrate that BMP-6 increases IL-1β expression in macrophages via the receptors ALK3 and BMPRII as well as the downstream signaling protein Smad1. Surprisingly though, inhibition of the ERK and JNK non-Smad pathways also completely blocked the induction of IL-1β by BMP-6 in macrophages. Further analysis revealed that a physical interaction between the transcription factor PU.1 and Smad 1 is necessary for the upregulation of IL-1β expression by BMP-6 in macrophages. Taken together, these results demonstrate that BMP-6-induced IL-1β expression in macrophages is mediated via a cross-talk between the Smad and the non-Smad pathways through Smad1 and PU.1.

Alkaline Phosphatase, Tartrate-resistant Acid Phosphatase, and BMP6(Vgr-1) Expression during Chondrocyte Differentiation and Osteogenesis in Fetal Mouse Metatarsals

Gu, Y.,Gitelman, S. E.,Martincic, D.,Zernik, J. H.,Minkin, C. Korean Academy of Oral Biology and the UCLA Dental 1997 International Journal of Oral Biology Vol.22 No.2

Changes in the expression of alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP or TrATPase, tartrate-resistant acid-ATPase), and bone morphogenic protein-6 (BMP6, also known as Vgr-1) were characterized during chondrocyte differentiation and osteogenesis in fetal mouse metatarsals. Histochemical observations demonstrate an increase in ALP expression both in differentiating hypertrophic chondrocytes and in osteogenic cells of the surrounding perichondrium/periosteum during days 15, 16, 17, and 18, of gestation. Expression of TRAP is first localized to a few small mononuclear osteoclast precursors in the periosteum of day 16 limbs and then a marked increase is demonstrated between days 17 and 18 of gestation as osteoclasts/chondroclasts invade periosteal bone surfaces and spicules of mineralized cartilage. Enzyme assays show a 9-fold increase in ALP activity in the developing limbs from day 15 to day 18 of gestation (0.05 to 0.43 mmole p-nitrophenol hydrolyzed/min/mg protein), and a 5.5-fold increase in TrATPase activity for the same time period (0.15 to 0.80% [^32P]g-ATP hydrolyzed/min/mg protein). Steady state levels of RNA for ALP, TRAP, and BMP6/Vgr-1 were evaluated using quantitative PCR amplification and our results demonstrate a 5-fold increase in ALP, a 500-fold increase in TRAP/TrATPase, and a 6-fold increase in BMP6/Vgr-1 mRNA levels between days 15 and 18 of gestation.