Expression and Purification of Biologically Active Human Bone Morphogenetic Protein-4 in Recombinant Chinese Hamster Ovary Cells

( Minyub Cha ),( Nara Han ),( Jia Pi ),( Yongsu Jeong ),( Kwanghee Baek ),( Jaeseung Yoon ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.7

Bone morphogenetic protein-4 (BMP-4) is considered to have therapeutic potential for various diseases, including cancers; however, the high expression of biologically active recombinant human BMP-4 (rhBMP-4) needed for its manufacture for therapeutic purposes has yet to be established. In the current study, we established a recombinant Chinese hamster ovary (rCHO) cell line overexpressing rhBMP-4 as well as a production process using 7.5-l bioreactor (5 L working volume). The expression of the mature rhBMP-4 was significantly enhanced by recombinant furin expression. The combination of a chemically defined medium and a nutrient supplement solution for high expression of rhBMP-4 was selected and used for bioreactor cultures. The 11-day fed-batch cultures of the established rhBMP-4-expressing rCHO cells in the 7.5-L bioreactor produced approximately 32 mg/l of rhBMP-4. The mature rhBMP-4 was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure, resulting in a recovery rate of approximately 55% and a protein purity greater than 95%. The N-terminal amino acid sequences and N-linked glycosylation of the purified rhBMP-4 were confirmed by N-terminal sequencing and de-N-glycosylation analysis, respectively. The mature purified rhBMP-4 has been proved to be functionally active, with an effective dose concentration of EC₅₀ of 2.93 ng/ml.

Overexpression of bone morphogenetic protein 4 in STO fibroblast feeder cells represses the proliferation of mouse embryonic stem cells in vitro

김구희,이공락,최형임,박능화,정헌택,한인섭 생화학분자생물학회 2012 Experimental and molecular medicine Vol.44 No.7

Embryonic stem cells (ESCs) can be propagated in vitro on feeder layers of mouse STO fibroblast cells. The STO cells secrete several cytokines that are essential for ESCs to maintain their undifferentiated state. In this study, we found significant growth inhibition of mouse ESCs (mESCs) cultured on STO cells infected with adenovirus containing a dominant-negative mutant form of IκB (rAd-dnIκB). This blockage of the NF-κB signal pathway in STO cells led to a significant decrease in [3H]thymidine incorporation and colony formation of mESCs. Expression profile of cytokines secreted from the STO cells revealed an increase in the bone morphogenetic protein4 (BMP4) transcript level in the STO cells infected with adenoviral vector encoding dominant negative IκB (rAd-dnIκB). These results suggested that the NF-κB signaling pathway represses expression of BMP4 in STO feeder cells. Conditioned medium from the rAd-dnIκB-infected STO cells also significantly reduced the colony size of mESCs. Addition of BMP4 prevented colony formation of mESCs cultured in the conditioned medium. Our finding suggested that an excess of BMP4 in the conditioned medium also inhibits proliferation of mESCs.

비침습식 미세전류자극이 토끼 경골의 골절 후 BMP-4 발현에 미치는 영향

조미숙(Cho mi-suk) 한국콘텐츠학회 2009 한국콘텐츠학회 종합학술대회 논문집 Vol.7 No.1

골절 후 골 유합에 대한 미세직류전류 자극의 효과를 관찰하기 위하여 체중 2.5~3 ㎏내외의 6개월 령 뉴질랜드 웅성 토끼 24마리를 대상으로 경골 골절 후 미세전류 자극을 적용한 실험군과 비적용군인 대조군으로 나누어 3일, 7일, 14일 및 28일 후 BMP-4에 대한 면역조직화학적 염색을 실행하여 다음과 같은 결론을 얻었다. BMP-4의 발현은 비침습식 미세전류를 적용한 실험군과 자연치유군인 대조군 모두 시간이 경과함에 따라 통계학적으로 유의하게 증가하다가 감소되었다. 그러나 실험군에서 경골 골절 3일 후 대조군은 하버씨계의 간질층판을 중심으로 약한 갈색의 면역양성반응(+)을 보였으나 실험군의 경우 중등도의 면역양성반응(++)을 보였다. 경골 골절 7일 후 대조군은 하버씨계의 동심원과 간질층판을 중심으로 중등도의 갈색의 면역양성반응(++)을 보였으나 실험군의 경우 바깥층판을 포함하여 매우 강한 갈색의 면역양성반응(++++)을 보였다. 경골 골절 14일 후 대조군은 하버씨계의 간질층판과 동심원을 중심으로 강한 갈색의 면역양성반응(+++)을 보였으며 실험군 또한 강한 면역양성반응(+++)을 보였다. 그 후 점차 감소하여 경골 골절 28일 후 대조군은 하버씨계의 간질층판을 중심으로 약한 갈색의 면역양성반응(+)을 보였으나 실험군의 경우 중등도의 면역양성반응(++)을 보였다. 위의 결과로 보면 골절 후 미세전류를 비침습적으로 적용할 때 치유과정 초기에 골형성단백질인 BMP-4의 발현을 증가시켜 골절 치유를 촉진시킴을 알 수 있다. This study aims to examine the effects of non-invasive constant micro current stimulation on expression of Bone Morphogenetic Protein(BMP) 4 after tibia fracture in rabbits. Twenty four rabbits with tibia fracture were randomly divided into control and experimental groups. Each group was divided into four subgroups, based on the duration of the experiment (3, 7, 14, 28 days). The experimental groups received a constant microcurrent stimulation of 20~25 ㎂ intensity with surface Ag-AgCl electrode (diameter 1㎝, Biopac, U.S.A.) for 24 hours a day. Cathode of the microcurrent stimulator located on the tibia directly. anode of it did on the gastrocnemius muscle Rabbits were sacrificed on each of the postoperative days 3, 7, 14, 28. To investigate how non- invasive constant microcurrent stimulation affects bone healing, immunohistochemical analysis of BMP-4 was performed at each point. After evaluation, the test results are as follows: Comparisons of immunohistochemical observation of BMP-4 in 7 days after tibial fracture show that there was shown to be a moderate positive reaction (++) on concentric circles of Harversian system and the interstitial lamella in the control group, while there was a very strong positive reaction (++++) on concentric circles of Harversian system and interstitial lamellain the experimental group. These results suggest that applying non-invasive constant microcurrent stimulation on fractured bone is helpful to bone healing.

The Role of Microtubule in the Regulation of Endochondral Bone Formation in the Developing Mouse Cranial Base

Hyuk-Jae Kwon(권혁제),Jong-Min Lee(이종민),Kyoung-Won Cho(조경원),Jeong-Oh Shin(신정오),Sung-Won Cho(조성원),Min-Jung Lee(이민정),Han-Sung Jung(정한성) 대한체질인류학회 2011 해부·생물인류학 (Anat Biol Anthropol) Vol.24 No.2

머리뼈바닥의 발생 중에 일어나는 연골속뼈발생은 다양한 세포현상과 연골바탕질 대사가 관여하는 매우 복잡한 과정이다. 연골세포에서는 증식, 형태변화, 증식 종료, 비대, 세포자멸사가 일어나고, 연골세포 주변으로는 연골바탕질이 활발하게 생성되었다가 제거되면서 뼈조직에 의해서 대체됨이 밝혀졌다. 그러나 이러한 복잡한 변화가 일어나는 동안 미세관이 어떠한 역할을 하는지에 대해서는 뚜렷하게 밝혀진 바가 없다. 그러므로 본 연구는 머리뼈바닥 발생 중 연골속뼈발생 동안의 미세관의 역할을 밝히고자 하였다. 미세관의 구성 단백질인 튜불린에 결합하는 약물 노코다졸을 사용하여 머리뼈바닥 조직을 기관배양 함으로써 미세관과 머리뼈바닥 발생의 관계를 조사하였다. 배양된 조직에서 일어나는 형태학적, 분자적 변화를 확인하기 위하여 헤마톡실린-에오신염색, 면역조직화학염색, 세포 개수 계산을 통한 분석이 이루어졌다. 나비뒤통수결합 부위 내의 미세관에 가해진 변화에 의해서 Ki67, 뼈발생표지자 BSP, BMP4의 발현이 감소하였다. 비대층 구역에서는 연골세포의 비대과정이 억제되었고, 뼈발생중심에서는 연골조직의 제거 및 뼈조직의 생성이 억제되었다. 본 연구는 미세관이 연골속뼈발생의 다양한 과정에 관여함을 밝혔다. 이러한 결과는 머리뼈 성장 및 머리뼈 바닥 기형과 관련된 질병을 이해하는 자료로 사용될 수 있을 것으로 사료된다. Endochondral bone formation of the developing cranial base is a complex process. This mechanism requires precise orchestration of many cellular events and cartilage matrix metabolism, such as proliferation, becoming round in shape, termination of proliferation, hypertrophic size-increase, and finally programmed cell death. Active formation and degradation of cartilage matrix take place, in which microtubules are involved for intracellular events; bone apposition follows these events. However, the involvement of microtubules during these changes in the developing cranial base has not been identified yet. Thus, we investigated the involvement of microtubules in the regulation of endochondral bone formation during cranial base development. Using tubulin-binding drug nocodazole, we examined the effects of altering the structure and function of microtubules during in vivo organ culture of the mouse cranial base. Cultured specimens were analyzed with HE staining, immunohistochemistry, and cell counting in order to study the morphological and molecular changes that occurred in the tissues. Disruption of the microtubular array by nocodazole reduced cells expressing proliferation marker Ki67, osteogenic marker BSP, and BMP4 within the sphenooccipital synchondrosis region; chondrocyte hypertrophy was ceased in the hypertrophic zone; degeneration of cartilage matrix and bone matrix apposition was inhibited in the ossification center of the basooccipital cranial base. Our data demonstrated that disruption of microtubules by nocodazole have multiple inhibitory effects on the sequential changes that occur during endochondral bone formation, suggesting the importance of normal microtubule-polymerization in cranial base development.

Bone morphogenetic protein 4 stimulates neuronal differentiation of neuronal stem cells through the ERK pathway

문병산,윤주용,Mi-Yeon Kim,이상훈,Thomas Choi,최강열 생화학분자생물학회 2009 Experimental and molecular medicine Vol.41 No.2

Bone morphogenic protein 4 (BMP4), a member of the TGF-β superfamily, induced neural differentiation of neural stem cells (NSCs) grown in a medium containing basic fibroblast growth factor (bFGF). The Ras protein level and the activities of the downstream ERKs were increased by transfection of BMP4 or treatment with recombinant BMP4. The effects of BMP4, including activation of the Ras-ERK pathway and induction of the neuron marker β-tubulin type III (Tuj1), were blocked by co-treatment of the BMP4 antagonist, noggin. The roles of the Ras-ERK pathway in neuronal differentiation by BMP4 were revealed by measuring the effect of the ERK pathway inhibition by dominant negative Ras or PD98059, the MEK specific inhibitor. BMP4 is a transcriptional target of Wnt/β-catenin signaling, and both the mRNA and protein levels of BMP4 were increased by treatment of valproic acid (VPA), a chemical inhibitor of glycogen synthase kinase 3β (GSK3β) activating the Wnt/β-catenin pathway. The BMP4- mimicking effects of VPA, activation of the Ras-ERK pathway and induction of Tuj1, also were blocked by noggin. These results indicate the potential therapeutic usage of VPA as a replacement for BMP4.

Nox4-Mediated Cell Signaling Regulates Differentiation and Survival of Neural Crest Stem Cells

이지은,조규은,이경은,김재상,배윤수 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.12

The function of reactive oxygen species (ROS) as second messengers in cell differentiation has been demonstrated only for a limited number of cell types. Here, we used a well-established protocol for BMP2-induced neuronal differentiation of neural crest stem cells (NCSCs) to examine the function of BMP2-induced ROS during the process. We first show that BMP2 indeed induces ROS generation in NCSCs and that blocking ROS generation by pretreatment of cells with diphenyleneiodonium (DPI) as NADPH oxidase (Nox) inhibitor inhibits neuronal differentiation. Among the ROS-generating Nox isozymes, only Nox4 was expressed at a detectable level in NCSCs. Nox4 appears to be critical for survival of NCSCs at least in vitro as down-regulation by RNA interference led to apoptotic response from NCSCs. Interestingly, development of neural crest-derived peripheral neural structures in Nox4-/- mouse appears to be grossly normal, although Nox4-/- embryos were born at a sub-Mendelian ratio and showed delayed over-all devel-opment. Specifically, cranial and dorsal root ganglia, derived from NCSCs, were clearly present in Nox4-/- embryo at embryonic days (E) 9.5 and 10.5. These results suggest that Nox4-mediated ROS generation likely plays important role in fate determination and differentiation of NCSCs, but other Nox isozymes play redundant function during embryogenesis.

Nox4-Mediated Cell Signaling Regulates Differentiation and Survival of Neural Crest Stem Cells

Lee, Ji-Eun,Cho, Kyu Eun,Lee, Kyung Eun,Kim, Jaesang,Bae, Yun Soo Korean Society for Molecular and Cellular Biology 2014 Molecules and cells Vol.37 No.12

The function of reactive oxygen species (ROS) as second messengers in cell differentiation has been demonstrated only for a limited number of cell types. Here, we used a well-established protocol for BMP2-induced neuronal differentiation of neural crest stem cells (NCSCs) to examine the function of BMP2-induced ROS during the process. We first show that BMP2 indeed induces ROS generation in NCSCs and that blocking ROS generation by pretreatment of cells with diphenyleneiodonium (DPI) as NADPH oxidase (Nox) inhibitor inhibits neuronal differentiation. Among the ROS-generating Nox isozymes, only Nox4 was expressed at a detectable level in NCSCs. Nox4 appears to be critical for survival of NCSCs at least in vitro as down-regulation by RNA interference led to apoptotic response from NCSCs. Interestingly, development of neural crest-derived peripheral neural structures in Nox4-/- mouse appears to be grossly normal, although Nox4-/- embryos were born at a sub-Mendelian ratio and showed delayed over-all development. Specifically, cranial and dorsal root ganglia, derived from NCSCs, were clearly present in Nox4-/- embryo at embryonic days (E) 9.5 and 10.5. These results suggest that Nox4-mediated ROS generation likely plays important role in fate determination and differentiation of NCSCs, but other Nox isozymes play redundant function during embryogenesis.

Modulation of Endothelial Bone Morphogenetic Protein Receptor Type 2 Activity by Vascular Endothelial Growth Factor Receptor 3 in Pulmonary Arterial Hypertension

Hwangbo, Cheol,Lee, Heon-Woo,Kang, Hyeseon,Ju, Hyekyung,Wiley, David S.,Papangeli, Irinna,Han, Jinah,Kim, Jun-Dae,Dunworth, William P.,Hu, Xiaoyue,Lee, Seyoung,El-Hely, Omar,Sofer, Avraham,Pak, Boryeo American Heart Association 2017 Circulation Vol.135 No.23

<P>CONCLUSIONS: Our findings identify VEGFR3 as a key regulator of endothelial BMPR2 signaling and a potential determinant of PAH penetrance in humans.</P>

Mechanisms of Vascular Calcification: The Pivotal Role of Pyruvate Dehydrogenase Kinase 4

임재찬,이인규 대한내분비학회 2016 Endocrinology and metabolism Vol.31 No.1

Vascular calcification, abnormal mineralization of the vessel wall, is frequently associated with aging, atherosclerosis, diabetes mellitus, and chronic kidney disease. Vascular calcification is a key risk factor for many adverse clinical outcomes, including ischemic cardiac events and subsequent cardiovascular mortality. Vascular calcification was long considered to be a passive degenerative process, but it is now recognized as an active and highly regulated process similar to bone formation. However, despite numerous studies on the pathogenesis of vascular calcification, the mechanisms driving this process remain poorly understood. Pyruvate dehydrogenase kinases (PDKs) play an important role in the regulation of cellular metabolism and mitochondrial function. Recent studies show that PDK4 is an attractive therapeutic target for the treatment of various metabolic diseases. In this review, we summarize our current knowledge regarding the mechanisms of vascular calcification and describe the role of PDK4 in the osteogenic differentiation of vascular smooth muscle cells and development of vascular calcification. Further studies aimed at understanding the molecular mechanisms of vascular calcification will be critical for the development of novel therapeutic strategies.

In Vitro Differentiation of Porcine Epiblast Stem Cells into Primordial Germ Cells through Embryoid Body Formation, BMP 4 and Retinoic Acid

Sang-Ki Baek,Tae-Suk Kim,Young-Soo Cho,Ik-Seong Kim,Sung-Woo Kim,Hyun Kim,Hwan-Hoo Seong,Joon-Hee Lee 한국동물생명공학회(구 한국동물번식학회) 2015 발생공학 국제심포지엄 및 학술대회 Vol.2015 No.10