Effect of combinatorial bone morphogenetic protein 2 and bone morphogenetic protein 7 gene delivery on osteoblastic differentiation

Young Bae,Kyoung-Hwa Kim,김수환,Chul-Woo Lee,구기태,김태일,설양조,Young Ku,류인철,정종평,이용무 대한치주과학회 2009 Journal of Periodontal & Implant Science Vol.39 No.2

Purpose: Gene therapy (ex vivo) has recently been used as a means of delivering bone morphogenetic proteins (BMPs) to sites of tissue regeneration. In the present study, we investigated the effect of co-transduction of adenoviruses expressing BMP-2 and BMP-7 on osteogenesisof C2C12 cells in vitro. Methods: A replication-defective human adenovirus 5 (Ad5) containing a cDNA for BMPs in the E1 region of the virus (Ad5BMP-2 and Ad5BMP-7) was constructed by in vivo homologous recombination. Functional activity of Ad5BMP-2 and Ad5BMP-7 were evaluated in mouse stromal cells (W20-17cells). C2C12 cells are transduced with various MOI (multiplicity of infection) of Ad5BMP-2 and Ad5BMP-7 to assess most effective and stable titer. Based on this result, C2C12 cells were transduced with Ad5BMP-2 and Ad5BMP-7 alone or by combination. BMPs expression, alkaline phosphatase (ALPase) activity, cell proliferation, and mineralization were assessed. Results: Ad5BMP-2 and Ad5BMP-7 are successfully transduced to W20-17 cells, and secreted BMPs stimulated cell differentiation. Also, C2C12 cells transduced with Ad5BMPs showed expression of BMPs and increased ALPaseactivity. In all groups, cell proliferation was observed over times. At 7days, cells co-transduced with Ad5BMP-2 and Ad5BMP-7 showed lower proliferation than the others. C2C12 cells co-transduced with Ad5BMP-2 and Ad5BMP-7 had greater ALPaseactivity than that would be predicted if effect of individual Ad5BMPs were additive. Little mineralized nodule formation was detected in cells transduced with individual Ad5BMPs. In contrast, Ad5BMP-2 and Ad5BMP-7 combination stimulated mineralization after culturing for 10 days in mineralizing medium. Conclusions: Present study demonstrated that adenoviruses expressing BMPs gene successfully produced BMPs protein and these BMPs stimulated cells to be differentiated into osteoblastic cells. In addition, the osteogenic activity of Ad5BMPs can be synergistically increased by co-transduction of cells with Ad5BMP-2 and Ad5BMP-7.(J Korean Acad Periodontol 2009;39:279-286)

Effect of combinatorial bone morphogenetic protein 2 and bone morphogenetic protein 7 gene delivery on osteoblastic differentiation

Bae, Young,Kim, Kyoung-Hwa,Kim, Su-Hwan,Lee, Chul-Woo,Koo, Ki-Tae,Kim, Tae-Il,Seol, Yang-Jo,Ku, Young,Rhyu, In-Chul,Chung, Chong-Pyoung,Lee, Yong-Moo Korean Academy of Periodontology 2009 Journal of Periodontal & Implant Science Vol.39 No.2

Purpose: Gene therapy (ex vivo) has recently been used as a means of delivering bone morphogenetic proteins (BMPs) to sites of tissue regeneration. In the present study, we investigated the effect of co-transduction of adenoviruses expressing BMP-2 and BMP-7 on osteogenesisof C2C12 cells in vitro. Methods: A replication-defective human adenovirus 5 (Ad5) containing a cDNA for BMPs in the E1 region of the virus (Ad5BMP-2 and Ad5BMP-7) was constructed by in vivo homologous recombination. Functional activity of Ad5BMP-2 and Ad5BMP-7 were evaluated in mouse stromal cells (W20-17cells). C2C12 cells are transduced with various MOI (multiplicity of infection) of Ad5BMP-2 and Ad5BMP-7 to assess most effective and stable titer. Based on this result, C2C12 cells were transduced with Ad5BMP-2 and Ad5BMP-7 alone or by combination. BMPs expression, alkaline phosphatase (ALPase) activity, cell proliferation, and mineralization were assessed. Results: Ad5BMP-2 and Ad5BMP-7 are successfully transduced to W20-17 cells, and secreted BMPs stimulated cell differentiation. Also, C2C12 cells transduced with Ad5BMPs showed expression of BMPs and increased ALPaseactivity. In all groups, cell proliferation was observed over times. At 7days, cells co-transduced with Ad5BMP-2 and Ad5BMP-7 showed lower proliferation than the others. C2C12 cells co-transduced with Ad5BMP-2 and Ad5BMP-7 had greater ALPaseactivity than that would be predicted if effect of individual Ad5BMPs were additive. Little mineralized nodule formation was detected in cells transduced with individual Ad5BMPs. In contrast, Ad5BMP-2 and Ad5BMP-7 combination stimulated mineralization after culturing for 10 days in mineralizing medium. Conclusions: Present study demonstrated that adenoviruses expressing BMPs gene successfully produced BMPs protein and these BMPs stimulated cells to be differentiated into osteoblastic cells. In addition, the osteogenic activity of Ad5BMPs can be synergistically increased by co-transduction of cells with Ad5BMP-2 and Ad5BMP-7.

Bone Morphogenic Protein-7의 복막섬유화 억제효과와 기전

서지연 ( Ji Yeon Seo ),하헌주 ( Hun Joo Ha ),유미라 ( Mi Ra Yu ),김재룡 ( Jae Ryong Kim ),안면환 ( Myun Whan Ahn ),이희발 ( Hi Bahl Lee ) 대한신장학회 2007 Kidney Research and Clinical Practice Vol.26 No.1

Purpose: Bone morphogenic protein (BMP)-7, a member of TGF-β1 superfamily, is an endogenous antifibrotic protein highly expressed in normal kidney. It is not known, however, whether human peritoneal mesothelial cells (HPMC) express BMP-7 or if BMP-7 protects against peritoneal fibrosis and by what mechanism. We examined the effect of BMP-7 overexpression in TGF-β1-induced epithelial-mesenchymal transition (EMT) of HPMC and in TGF-β1 signaling in HPMC to elucidate the mechanisms of antifibrotic effect of BMP-7. Methods: Growth arrested and synchronized HPMC were stimulated with 2 ng/mL of TGF-β1 to induce EMT. HPMC were transiently transfected with adenovirus-mediated human BMP-7 (AdBMP-7) or with GFP (AdGFP). EMT was defined as downregulation of E-cadherin and upregulation of α-smooth muscle actin (SMA). Results: HPMC constitutively expressed BMP-7 mRNA and protein. BMP-7 mRNA and protein expression were significantly inhibited by 50 mM D-glucose, 2x diluted commercial peritoneal dialysis solution, and 2 ng/ml of TGF-β1. Transfection of AdBMP-7 resulted in 2.5-fold increase in BMP-7 mRNA expression in HPMC. TGF-β1 significantly decreased E-cadherin and increased α-SMA expression in GFP transfected cells. BMP-7 overexpression effectively reversed TGF-β1-induced E-cadherin and α-SMA expression and significantly suppressed TGF-β1-induced phosphorylation of Smad2/3, ERK1/2, JNK, and p38 MAPK in HPMC as compared to GFP transfected cells. Conclusion: BMP-7 is an endogenous antifibrotic protein and downregulation of BMP-7 in HPMC by high glucose, PD solution, and TGF-β1 may permit the development of peritoneal fibrosis during long-term PD. Our data demonstrate that BMP-7 overexpression reverses TGF-β1-induced EMT of HPMC and consequent peritoneal fibrosis possibly through inhibition of Smad2/3 and MAPK phosphorylation.

고초균을 이용한 재조합 인간 골 형성 단백질-7의 발현과 정제

김춘광(Chun-Kwang Kim),오성덕(Sung-Duk Oh),이종일(Jong Il Rhee) 한국생물공학회 2010 KSBB Journal Vol.25 No.3

Bone morphogenetic protein-7 (BMP-7) is one of important growth factors for skeletal development and bone growth. In this work, BMP-7 was efficiently expressed in recombinant Bacillus subtilis. The mature BMP-7 protein indicated molecular weight of 15.4 kDa by Western blot assay and was secreted into culture medium with 0.35 ng/mL. The extracellular and intracellular rhBMP-7 proteins were purified by using a FPLC system with an ion exchange column and a gel filtration column. The extracellular and intracellular rhBMP-7 proteins had finally a 57.1% purity and a 36.2% purity, respectively. The purified rhBMP-7 proteins showed an intact biological activity which stimulated alkaline phophatase (ALP) activity in MC3T3-E1 cells.

Adenovirus Mediated Human BMP-7 Gene Transfer Induces Osteogenic Differentiation of Murine Mesenchymal Progenitor Cell Line C3H10T1/2

이동희,양형철,박종철,김나령 한국생체재료학회 2006 생체재료학회지 Vol.10 No.1

Osteogenic differentiation of a mesenchymal progenitor stimulated by BMP-7 was studied. A recombinant adenovirus with hBMP-7 gene was constructed and this virus successfully mediated the gene transfer and BMP-7 protein expression in the mesenchymal progenitor cell line C3H10T1/2. Our in vitro study demonstrated that BMP-7 gene transfer and expression, mediated by adenovirus, induced the proliferation and differentiation of C3H10T1/2 cells. This cell proliferation is dependent on adenovirus BMP-7 dose (pfu) and directly related to the number of BMP genes transferred. Our results demonstrated that adenovirus-mediated BMP-7 gene transfer into the cells could have an effect similar to that of recombinant hBMP-7 protein on cell differentiation. Moreover, gene transfer has the advantage of directing the cell to continuously produce BMP-7 protein. Therefore, these findings can potentially be utilized clinically to stimulate fracture repair and heal bone defects.

Expression and Purification of Recombinant Human Bone Morphogenetic Protein-7 (rhBMP-7) in Bacillus subtilis

Chun-Kwang Kim,오성덕,Jong Il Rhee,Esther Meerim Lee,윤택림 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.5

The polypeptide representing the mature part of human bone morphogenetic protein-7 (BMP-7) was cloned and efficiently expressed in Bacillus subtilis. Recombinant B. subtilis had a clear band for rhBMP-7, a homodimeric protein with an apparent molecular weight of 15.4 kDa and produced 350 pg rhBMP-7/mL of culture medium. The extracellular and intracellular rhBMP-7 was purified in two steps using a fast performance liquid chromatography (FPLC) system with an ion-exchange column and a gel filtration column. The extracellular rhBMP-7 had a purity of 57.1% and a yield of 58.8%, while the purity of the intracellular rhBMP-7 was 36.2% with a yield of 51.4%. The rhBMP-7 produced in this work also stimulated alkaline phosphatase (ALP) activity in a dose-dependent manner,i.e. 2.5- and 8.9-fold at 100 and 300 ng rhBMP-7/mL,respectively, and showed intact biological activity.

복막투석 동물모델에서 Adenoviral vector를 이용한 Bone Morphogenic Protein-7 유전자 치료가 복막섬유화에 미치는 영향

최지영 ( Ji Young Choi ),진미경 ( Mi Kyung Jin ),전주현 ( Joo Hyun Chun ),현승혜 ( Seung Hyea Hyun ),최희정 ( Hee Jeong Choi ),최혁준 ( Hyuk Joon Choi ),조지형 ( Ji Hyung Cho ),김미형 ( Mi Hyung Kim ),류혜명 ( Hye Myung Ryu ),오 대한신장학회 2008 Kidney Research and Clinical Practice Vol.27 No.4

Purpose: TGF-β-induced epithelial-mesenchymal transition (EMT) is associated with peritoneal fibrosis during PD. We conducted this study to evaluate the effect of BMP-7 adenoviral gene transfer on the functional and structural changes of peritoneum and whether it is associated with peritoneal EMT using an animal PD model. Methods: Forty Sprague-Dawley rats were divided into five groups; Control (C, n=8), Dialysis (D, n= 8), Rest (R, n=8), BMP-7 (B, n=8) and LacZ (L, n=8) group. Peritoneal function was assessed on baseline, 3rd, 6th, 8th weeks after PD. Immunohistochemistry for TGF-β, VEGF, laminin and aquaporin-1 was performed in addition to morphometric analysis of peritoneum. Immunofluorescence staining with western blotting for α-SMA and E-cadherin, as markers of EMT, was performed. Results: The thickness of submesothelial matrix was highest in D and significantly decreased in B compared to D, R and L. D/D0 glucose at 8 weeks was significantly increased in B and L compared to that of at 6 weeks, but there were no significant differences among R, B and L at 8 weeks. TGF-β1 and VEGF expression was observed in submesothelial matrix in D and decreased in R, B and L. Peritoneal fibrosis and functional deterioration of peritoneal membrane were associated with EMT, which was partially reversed in R, B and L. Conclusions: BMP-7 gene transfer to peritoneum was not associated with the additive therapeutic effect on peritoneal function compared to the peritoneal rest, although it improved morphologic changes of peritoneum.

The Effect of Protein Transduction Domain Recombinant Bone Morphogenetic Protein-7 on Epithelial-Mesenchymal Transition in Peritoneal Mesothelial Cells

JIMIN PARK,Shin-Wook Kang 한국실험동물학회 2016 한국실험동물학회 학술발표대회 논문집 Vol.2016 No.08

Use of stem-cell sheets expressing bone morphogenetic protein-7 in the management of a nonunion radial fracture in a Toy Poodle

송재용,김용선,권오경,강병재 대한수의학회 2017 Journal of Veterinary Science Vol.18 No.4

A 12-year-old castrated Toy Poodle was referred to the Kangwon National University Animal Hospital with an oligotrophic nonunion fracture in the distal 1/3 of the left radius and an intact ulna. After fixation by a locking plate and screws, adipose-derived mesenchymal stem-cell sheets expressing bone morphogenetic protein 7 (BMP-7) were transplanted to the fracture site to enhance the healing activity. The fracture was healed at 9 weeks after surgery. In the present case, the mesenchymal stem-cell sheets expressing BMP-7 promoted bone regeneration and healing in a nonunion fracture.

Sulforaphane Ameliorates Diabetes-Induced Renal Fibrosis through Epigenetic Up-Regulation of BMP-7

Lili Kong,Hongyue Wang,Chenhao Li,Huiyan Cheng,Yan Cui,Li Liu,Ying Zhao 대한당뇨병학회 2021 Diabetes and Metabolism Journal Vol.45 No.6

Background: The dietary agent sulforaphane (SFN) has been reported to reduce diabetes-induced renal fibrosis, as well as inhibit histone deacetylase (HDAC) activity. Bone morphologic protein 7 (BMP-7) has been shown to reduce renal fibrosis induced by transforming growth factor-beta1. The aim of this study was to investigate the epigenetic effect of SFN on BMP-7 expression in diabetes-induced renal fibrosis.Methods: Streptozotocin (STZ)-induced diabetic mice and age-matched controls were subcutaneously injected with SFN or vehicle for 4 months to measure the in vivo effects of SFN on the kidneys. The human renal proximal tubular (HK11) cell line was used to mimic diabetic conditions in vitro. HK11 cells were transfected to over-express HDAC2 and treated with high glucose/palmitate (HG/Pal) to explore the epigenetic modulation of BMP-7 in SFN-mediated protection against HG/Pal-induced renal fibrosis.Results: SFN significantly attenuated diabetes-induced renal fibrosis in vivo. Among all of the HDACs we detected, HDAC2 activity was markedly elevated in the STZ-induced diabetic kidneys and HG/Pal-treated HK11 cells. SFN inhibited the diabetes-induced increase in HDAC2 activity which was associated with histone acetylation and transcriptional activation of the BMP-7 promoter. HDAC2 over-expression reduced BMP-7 expression and abolished the SFN-mediated protection against HG/Pal-induced fibrosis in vitro.Conclusion: Our study demonstrates that the HDAC inhibitor SFN protects against diabetes-induced renal fibrosis through epigenetic up-regulation of BMP-7.