[논문]심근세포로의 분화에 관여하는 새로운 생리활성 단백질 SPP2의 발굴

전세진

doi:10.5352/jls.2023.33.1.64

심근세포로의 분화에 관여하는 새로운 생리활성 단백질 SPP2의 발굴
Identification and Characterization of Secreted Phosphoprotein 2 as a Novel Bioactive Protein for Myocardial Differentiation 원문보기

생명과학회지 = Journal of life science, v.33 no.1, 2023년, pp.64 - 72

전세진 (안동대학교 생명공학부 생명백신공학전공)

초록
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심장 발생과정에 관여하는 주요 전사인자들의 기능에 대한 규명 등의 발전에도 불구하고 줄기 세포에서 매우 효율적인 심근 세포로의 분화를 촉진하는 새로운 생체 활성 분자를 찾는 것이 여전히 필요하다. 마우스배아줄기세포(mESC) 유래 심근세포의 Illumina 발현 마이크로어레이 데이터를 분석하였다. 미분화 mESCs와 비교하여 mESC 유래 심근세포에서 4배 이상 유전자 발현이 증가한 276개 유전자가 스크리닝되었다. Secreted phosphoprotein 2 (Spp2)는 후보물질 중 하나이며 bone morphogenetic protein 2 (BMP2)에 대한 슈도수용체로서 BMP2 신호 전달을 억제하는 것으로 알려져 있다. 그러나 심근 형성과의 연관성은 알려진 바 없다. 우리는 mESC 세포주인 TC-1/Kh2와 E14를 이용하여 기능성 심근세포로 분화하는 동안 Spp2 발현이 증가함을 검증하였다. 흥미롭게도, Spp2 분비는 배아체(embryoid body, EBs) 형성 후 3일차에 일시적으로 증가했는데, 이는 Spp2의 분비가 ESCs의 심근세포로의 분화에 관여함을 시사한다. Spp2의 기능을 분석하기 위해, 우리는 BMP2를 처리하면 분화 경로를 근모세포에서 골모세포로 전환되는 특성을 가진 C2C12 마우스 근모세포 세포주를 사용하여 실험을 수행하였다. mESCs의 분화와 유사하게, Spp2의 전사는 C2C12 근모세포가 근관으로 분화됨에 따라 증가하였다. 특히, 분화 초기 단계에서 Spp2의 세포외 분비가 극적으로 증가하였다. 또한, Spp2-Flag 재조합 단백질로 처리하면 C2C12 근모세포의 근관으로의 분화가 촉진되었다. 종합하면, ESCs를 심근 세포로 분화시키는 새로운 생체 활성 단백질로 Spp2를 제안한다. 이것은 심근형성의 분자 경로를 이해하고 허혈성 심장질환에 대한 줄기세포 요법의 실험적 또는 임상적 발전을 촉진하는 역할을 할 것으로 기대한다.

Abstract ▼ AI-Helper

Despite several advances in identification of cardiac transcription factors, there are still needs to find new bioactive molecules that promote cardiomyogenesis from stem cells to highly efficient myocardial differentiation. We analyzed Illumina expression microarray data of mouse embryonic stem cells (mESCs)-derived cardiomyocytes. 276 genes were upregulated (≥ 4fold) in mESCs-derived cardiomyocytes compared undifferentiated ESCs. Secreted phosphoprotein 2 (Spp2) is one of candidates and is known to inhibit bone morphogenetic protein 2 (BMP2) signal transduction as a pseudoreceptor for BMP2. However, its function in cardiomyogenesis is unknown. We confirmed that Spp2 expression increased during the differentiation into functional cardiomyocytes using mESCs, TC-1/Kh2 and E14. Interestingly, Spp2 secretion transiently increased 3 days after formation of embryoid bodies (EBs), indicating that the extracellular secretion of Spp2 is involved in the differentiation of ESCs into cardiomyocytes. To characterize Spp2, we performed experiments using the C2C12 mouse myoblast cell line, which has the property of shifting the differentiation pathway from myoblastic to osteoblastic by treatment with BMP2. Similar to the differentiation of ESCs, transcription of Spp2 increased as C2C12 myoblasts differentiated into myotubes. In particular, Spp2 secretion increased dramatically in the early stage of differentiation. Furthermore, treatment with Spp2-Flag recombinant protein promoted the differentiation of C2C12 myoblasts into myotubes. Taken together, we suggest a novel bioactive protein Spp2 that differentiates ESCs into cardiomyocytes. This may be useful for understanding the molecular pathways of cardiomyogenesis and for experimental or clinical promotion of stem cell therapy for ischemic heart diseases.

주제어

표/그림 (5)

그림 Fig. 1. Expression of EGFP under the transcriptional control of alpha-MHC promoter in differentiating EBs and schematic presentation of the in vitro differentiation protocol of embryonic stem cells into cardiomyocytes. (A) A DNA construct is containing 1.8 kb promoter sequence of the mouse alpha-MHC gene between the EcoRI and SalI sites of pEGFP-1 (Clontech Laboratories, Inc., Palo Alto, CA). (B) EBs expressed EGFP only when they had been differentiated into cardiomyocytes after 10 days. Cell morphology was detected under an inverted phase contrast microscope (x200). Each image is representative of at least three independent experiments. (C-D) beating scoring standard (C) and GFP-positive scoring standard (D) of differentiated embryoid bodies and cardiomyocyte differentiation effect of cardiogenol C (CgC). Data are expressed as the mean ± S.E.M (n=4). The statistical analyses were conducted using analysis of variance (2-way Dunnett's multiple comparisons test) between groups (*; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001).
그림 Fig. 2. Global gene expression profiles in TC-1/Kh2 mESCs-derived cardiomyocytes differentiated by EB (embryoid body) formation. (A) Hierarchical clustering of probe sets as upregulated or downregulated in mESCs-derived cardiomyocytes with an expression level at least 2-fold higher or lower than in undifferentiated mESCs. Each probe set is represented by a single row of colored boxes; each array is represented by a single column. Rectangles corresponding to intermediately expressed probe sets are colored black, upregulated probe sets are indicated with red of increasing intensity, and weakly expressed probe sets with green of increasing intensity. The dendrogram on the left of the figure represents the similarity matrix of probe sets. (B) Differentiated cardiomyocyte exhibit both distinct and shared gene expression profiles. (C-F) Verification of marker gene transcriptional levels by real-time qPCR. Among them, Nanog, stemness marker (C), Nkx2.5, cardiac transcription factor (D), TnnT2, functional cardiomyocyte marker (E) and Spp2 (F). Data are expressed as the mean ± S.E.M (n=6). The statistical analyses were conducted using analysis of variance (2-way Dunnett's multiple comparisons test) between groups (*; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001).
그림 Fig. 3. Verification of expression patterns of Spp2 in cardiac differentiation from E14 mESCs. Development of contracting EBs over time in differentiating cultures of E14 mESCs. (A-D) Verification of marker gene transcriptional levels by real-time qPCR. Among them, Nanog, stemness marker (A), Nkx2.5, cardiac transcription factor (B), TnnT2, functional cardiomyocyte marker (C) and Spp2 (D). (E) Observation of the expression patterns of Spp2 in mESCs-derived cardiomyocytes during differentiating day 8 by Western blot analysis. Each image is representative of at least three independent experiments. Data are expressed as the mean ± S.E.M (n=6). The statistical analyses were conducted using analysis of variance (2-way Dunnett's multiple comparisons test) between groups (*; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001).
그림 Fig. 4. Confirmation of the expression patterns of Spp2 in myogenic differentiated C2C12 myoblasts. (A) Development of contracting C2C12 myotubes over time in differentiating cultures of C2C12 myoblasts. Each image is representative of at least three independent experiments. (B-C) Observation of the expression levels in undifferentiated C2C12 myoblasts and in differentiated C2C12 myotubes during days 7 by real-time qPCR (B), and Western blots (C). Each image is representative of at least three independent experiments. Data are expressed as the mean ± S.E.M (n=3-8). The statistical analyses were conducted using analysis of variance (2-way Dunnett's multiple comparisons test) between groups (*; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001).
그림 Fig. 5. Treatment with Spp2-Flag recombinant protein in myogenic differentiated C2C12 myoblasts. (A) Construct of expression vector, pCMV-Spp2-Flag and Western blots. Each image is representative of at least three independent experiments. (B) Development of contracting C2C12 myotubes over time in differentiating cultures of C2C12 myoblasts by treatment with 3 μM of CgC, 100μg/ml of recombinant protein Spp2-Flag. Each image is representative of at least three independent experiments. (C) Observation of the marker gene expression levels in differentiated C2C12 myotubes after 5 days by real-time qPCR with and without recombinant protein Spp2-Flag treatment. Data are expressed as the mean ± S.E.M (n=6). The two-tailed Mann-Whitney (MW) U-test was used for comparisons between two groups (*; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001).

AI 본문요약
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대상 데이터

감마조사 후 배아줄기세포주의 피더(feeder) 세포로 사용한다. TC-1/KH2 마우스배아줄기세포주(mouse embryonic stem cells; mESCs)는 alpha-myosin heavy chain (a-MHC) 유전자 프로모터에 enhanced green fluorescence protein (EGFP)가 클로닝된 벡터에 의해 형질전환된 마우스배아줄기세포주로 생명공학연구원의 남기환박사님에게서 제공받았다. 벡터 DNA는 Dr.
벡터 DNA는 Dr. Yamashita June (Kyoto University, Japan) 에게서 제공받았다(Fig. 1A).

데이터처리

2개의 그룹을 비교할 때에는 two-tailed Mann-Whitney (MW) U-test를 진행하고, 3개 이상의 그룹의 데이터를 비교할 때에는 one-way ANOVA 또는 2-way ANOVA (Dunnett's multiple comparisons test)를 진행한다
2차 항체는 goat anti-rabbit IgG Antibody, (H + L) horseradish peroxidase (HRP) conjugate 또는 rabbit anti-goat IgG Antibody, HRP conjugate를 이용하여 1: 3,000으로 TBST 용액에 희석하여 상온에서 1시간 부드럽게 흔들면서 반응시킨다. 면역반응 검출은 ECL Western Blot reagents (GE Healthcare Life Sciences, RPN 2106)을 이용하여, ChemiDoc XRS+ (BioRad)에서 검출하여 ImageLab Software (v6.1, BioRad)와 ImageJ (v1.53c, NIH)를 이용하여 정량화한다. 상대적인 정량을 위해 house-keeping protein β-Actin 또는 GAPDH를 함께 진행한다.
통계 분석은 GraphPad InStat software를 사용한다. 2개의 그룹을 비교할 때에는 two-tailed Mann-Whitney (MW) U-test를 진행하고, 3개 이상의 그룹의 데이터를 비교할 때에는 one-way ANOVA 또는 2-way ANOVA (Dunnett's multiple comparisons test)를 진행한다.

성능/효과

마커 유전자의 전사정도를 관찰한 결과, Nanog는 분화이후 지속적으로 발현이 감소하였으며(p<0.0001) (Fig
3D). 배아줄기세포의 심근세포로의 분화에 따른 Spp2의 발현 증가를 검증함으로써, Spp2가 심근세포로의 분화과정에 관여할 가능성을 제시한다.

후속연구

형질전환된 배아줄기세포주가 아닌 다른 세포주에서도 Spp2의 발현양상을 검증할 필요가 있었다. 다른 마우스 배아줄기세포주인 E14TG2a (E14) mESCs도 역시 배아체 형성 방식으로 심근세포로의 분화를 유도하였다.

참고문헌 (28)

Behfar, A., Zingman, L. V., Hodgson, D. M., Rauzier, J. M., Kane, G. C., Terzic, A. and Puceat, M. 2002. Stem cell differentiation requires a paracrine pathway in the heart. FASEB J. 16, 1558-1566.

상세보기
Beqqali, A., Kloots, J., Ward-van, Oostwaard D., Mummery, C. and Passier, R. 2006. Genome-wide transcriptional profiling of human embryonic stem cells differentiating to cardiomyocytes. Stem Cells 24, 1956-1967.

상세보기
Boheler, K. R., Czyz, J., Tweedie, D., Yang, H. T., Anisimov, S.V. and Wobus A. M. 2002. Differentiation of pluripotent embryonic stem cells into cardiomyocytes. Circ. Res. 91, 189-201.

상세보기
Brochmann, E. J., Behnam, K. and Murray, S. S. 2009. Bone morphogenetic protein-2 activity is regulated by secreted phosphoprotein-24 kd, an extracellular pseudoreceptor, the gene for which maps to a region of the human genome important for bone quality. Metabolism 58, 644-650.

상세보기
Brochmann, E. J., Simon, R. J., Jawien, J., Behnam, K., Sintuu, C., Wang, J. C. and Murray, S. S. 2010. Carboxy terminus of secreted phosphoprotein-24 kDa (spp24) is essential for full inhibition of BMP-2 activity. J. Orthop. Res. 28, 1200-1207.

상세보기
Dib, N., Michler, R. E., Pagani, F. D., Wright, S., Kereiakes, D. J., Lengerich, R., Binkley, P., Buchele, D., Anand, I., Swingen, C., Di, Carli, M. F., Thomas, J. D., Jaber, W. A., Opie, S. R., Campbell, A., McCarthy, P., Yeager, M., Dilsizian, V., Griffith, B. P., Korn, R., Kreuger, S. K., Ghazoul, M., MacLellan, W. R., Fonarow, G., Eisen, H. J., Dinsmore, J. and Diethrich, E. 2005. Safety and feasibility of autologous myoblast transplantation in patients with ischemic cardiomyopathy: four-year follow-up. Circulation 112, 1748-1755.

상세보기
Ding, S. and Schultz, P. G. 2004. A role for chemistry in stem cell biology. Nat. Biotechnol. 22, 833-840.

상세보기
Doss, M. X., Koehler, C. I., Gissel, C., Hescheler, J. and Sachinidis, A. 2004. Embryonic stem cells: a promising tool for cell replacement therapy. J. Cell. Mol. Med. 8, 465-473.

상세보기
Gonzalez, A., Rota, M., Nurzynska, D., Misao, Y., Tillmanns, J., Ojaimi, C., Padin-Iruegas, M. E., Muller, P., Esposito, G., Bearzi, C., Vitale, S., Dawn, B., Sanganalmath, S. K., Baker, M., Hintze, T. H., Bolli, R., Urbanek, K., Hosoda, T., Anversa, P., Kajstura, J. and Leri, A. 2008. Activation of cardiac progenitor cells reverses the failing heart senescent phenotype and prolongs lifespan. Circ. Res. 102, 597-606.

상세보기
Jacob, G. S., Daniel, R. B., Julie, A. B., Travis, H., Bingruo, W., Thomas, C. T., Stephen, E. H., Bin, Z., Yuji, M. and Yukiko, S. 2017. BMP2 expression in the endocardial lineage is required for AV endocardial cushion maturation and remodeling. Dev. Biol. 430, 113-128.

상세보기
Jamali, M., Rogerson, P. J., Wilton, S. and Skerjanc, I. S. 2001. Nkx2-5 activity is essential for cardiomyogenesis. J. Biol. Chem. 276, 42252-42258.

상세보기
Juan, F. S., Hristina, O. and Jelena, K. 2021. BMP2 downregulates urokinase-type plasminogen activator via p38 MAPK: Implications in C2C12 cells myogenic differentiation. Acta Histochem. 123, 151774.

상세보기
Karamboulas, C., Dakubo, G. D., Liu, J., De, Repentigny Y., Yutzey, K., Wallace, V. A., Kothary, R. and Skerjanc, I. S. 2006. Disruption of MEF2 activity in cardiomyoblasts inhibits cardiomyogenesis. J. Cell Sci. 119, 4315-4321.

상세보기
Kawai, T., Takahashi, T., Esaki, M., Ushikoshi, H., Nagano, S., Fujiwara, H. and Kosai, K. 2004. Efficient cardiomyogenic differentiation of embryonic stem cell by fibroblast growth factor 2 and bone morphogenetic protein 2. Circ. J. 68, 691-702.

상세보기
Keller, G. M. 1995. In vitro differentiation of embryonic stem cells. Curr. Opin. Cell Biol. 7, 862-869.

상세보기
Menasche, P., Alfieri, O., Janssens, S., McKenna, W., Reichenspurner, H., Trinquart, L., Vilquin, J. T., Marolleau, J. P., Seymour, B., Larghero, J., Lake, S., Chatellier, G., Solomon, S., Desnos, M. and Hagege, A. A. 2008. The Myoblast Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) trial: first randomized placebo-controlled study of myoblast transplantation. Circulation 117, 1189-1200.

상세보기
Mike, A. K., Koenig, X., Koley, M., Heher, P., Wahl, G., Rubi, L., Schnurch, M., Mihovilovic, M. D., Weitzer, G. and Hilber, K. 2014. Small molecule cardiogenol C upregulates cardiac markers and induces cardiac functional properties in lineage-committed progenitor cells. Cell Physiol. Biochem. 33, 205-221.

상세보기
Murray, E. J., Murray, S. S., Simon, R. and Behnam, K. 2007. Recombinant expression, isolation, and proteolysis of extracellular matrix-secreted phosphoprotein-24 kDa. Connect Tissue Res. 48, 292-299.

상세보기
Murray, S. S., Wang, J. C., Duarte, M. E., Zhao, K. W., Tian, H., Francis, T. and Brochmann Murray, E. J. 2015. The bone matrix protein secreted phosphoprotein 24 kD (Spp24): bone metabolism regulator and starting material for biotherapeutic materials. Histol. Histopathol. 30, 531-537.

상세보기
Oh, H., Bradfute, S. B., Gallardo, T. D., Nakamura, T., Gaussin, V., Mishina, Y., Pocius, J., Michael, L. H., Behringer, R. R., Garry, D. J., Entman, M. L. and Schneider, M. D. 2003. Cardiac progenitor cells from adult myocardium: Homing, differentiation, and fusion after infarction. Proc. Natl. Acad. Sci. USA. 100, 12313-12318.

상세보기
Oh, S. W., Lee, J. B., Kim, B., Jeon, S., Kim, M. K., Nam, K. H., Ha, J. R., Bhatia, M., Oh, G. T. and Kim, D. Y. 2012. Peptidomimetic small-molecule compounds promoting cardiogenesis of stem cells. Arch. Pharm. Res. 35, 1979-1988.

원문보기 상세보기
Oh, S. W., Kim, B., Jeon, S., Go, D. M., Kim, M. K., Baek, K., Oh, G. T. and Kim, D. Y. 2013. Identification and characterization of CW108F, a novel β-carboline compound that promotes cardiomyogenesis of stem cells. Life Sci. 93, 409-15.

상세보기
Ooi, O. C., Al Habib, H. F., Almsherqi, Z. A. and El Oakley, R. M. 2006. Stem cell transplantation: potential impact on heart failure. Cell Tissue Bank 7, 307-317.

상세보기
Sintuu, C., Murray, S. S., Behnam, K., Simon, R., Jawien, J., Silva, J. D., Duarte, M. E. and Brochmann, E. J. 2008. Full-length bovine spp24 [spp24 (24-203)] inhibits BMP-2 induced bone formation. J. Orthop. Res. 26, 753-758.

상세보기
Turner, M. E., White, C. A., Taylor, S. M., Neville, K., Rees-Milton, K., Hopman, W. M., Adams, M. A., Anastas siades, T. and Holden, R. M. 2021. Secreted phosphoprotein 24 is a biomarker of mineral metabolism. Calcif Tissue Int. 108, 354-363.

상세보기
Wu, X., Ding, S., Ding, Q., Gray, N. S. and Schultz, P. G. 2004. Small molecules that induce cardiomyogenesis in embryonic stem cells. J. Am. Chem. Soc. 126, 1590-1591.

상세보기
Yamamoto, N., Akiyama, S., Katagiri, T., Namiki, M., Kurokawa, T. and Suda, T. 1997. Smad1 and smad5 act downstream of intracellular signalings of BMP-2 that inhibits myogenic differentiation and induces osteoblast differentiation in C2C12 myoblasts. Biochem. Biophys. Res. Commun. 238, 574-580.

상세보기
Yuasa, S., Itabashi, Y., Koshimizu, U., Tanaka, T., Sugimura, K., Kinoshita, M., Hattori, F., Fukami, S., Shimazaki, T., Ogawa, S., Okano, H. and Fukuda, K. 2005. Transient inhibition of BMP signaling by Noggin induces cardiomyocyte differentiation of mouse embryonic stem cells. Nat. Biotechnol. 23, 607-611.

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표제어: PCR

동의어: Packet Collision Rate

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용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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저장형식	Text(ASCII format) Excel format RefWorks Direct Export RIS format (for Reference Manager, ProCite, EndNote), Scholar's Aids, Mendeley
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

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