Parathyroid hormone-related protein sub-gene regulates precartilagenous stem cells

doi:10.3969/j.issn.2095-4344.2013.10.017

Abstract

Abstract:

BACKGROUND: The interaction between parathyroid hormone-related protein and Indian hedgehog protein both can regulate the differentiation, maturation and proliferation of cartilage in epiphyseal zone, which can help the cells in the epiphyseal stay in the relatively stable state.
OBJECTIVE: To analyze the regulatory effects of parathyroid hormone-related protein sub-gene (1-36), (38-94) and (107-139) on precartilaginous stem cells and the relationship between parathyroid hormone-related protein (107-139) and other regulating factors.
METHODS: The pTRE-parathyroid hormone-related protein (1-36), pTRE-parathyroid hormone-related protein P(38-94) and pTRE-parathyroid hormone-related protein (107-139) were transfected into pTet-on precartilagenous stem cells by liposome transfection method. The cells were cultured by differentiation-induced medium and the Tet-on was induced by 1 mg/L doxycycline respectively. Then the changes of expression of proliferating cell nuclear antigen gene were examined by reverse transcription-PCR. Plasmids pTRE-parathyroid hormone-related protein (107-139) were transfected into pTet-on precartilagenous stem cells which were induced by doxycycline with different concentrations respectively. Then the changes of expression of genes such as collagen type Ⅱ, collagen type Ⅹ, Indian hedgehog protein, Ptc, Sox-9 and bone morphogenetic protein 6 were examined by semiquantitative reverse transcription-PCR.
RESULTS AND CONCLUSION: Compared with the group transfected with pTRE-parathyroid hormone-related protein (1-36) and pTRE-parathyroid hormone-related protein (38-94), the group transfected with pTRE- parathyroid hormone-related protein (107-139) expressed more proliferating cell nuclear antigen. For the group transfected with pTRE-parathyroid hormone-related protein (107-139), the expressions of collagen type Ⅱ and Sox9 were significantly increased, while the expression of Indian hedgehog protein did not change, and the expressions of Ptc, collagen type X and bone morphogenetic protein 6 were decreased. The expression of the genes showed a dose-dependent manner. As parts of parathyroid hormone-related protein, parathyroid hormone-related protein (107-139) may promote the proliferation and inhibit the differentiation of precartilagenous stem cells by regulating the expression of Sox-9, Ptc and bone morphogenetic protein 6. However, parathyroid hormone-related protein (1-36) and (38-94) were useless in proliferation of precancerous stem cells. pTet-on system can regulate the expression of alien genes effectively.

Key words: stem cells, stem cell culture and differentiation, parathyroid hormone-related protein, Indian hedgehog protein, precartilaginous stem cells, proliferation, differentiation, proliferating cell nuclear antigen, other grants-supported paper, stem cell photographs-containing paper

CLC Number:

R318

Zhang Shu-wei, Jin Wei, Zhu Shao-bo, Cheng Hao, Jin Lin, Tao Sheng-xiang. Parathyroid hormone-related protein sub-gene regulates precartilagenous stem cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(10): 1814-1820.

Figures/Tables 7

References

[1] Chau M,Forcinito P,Andrade AC,et al.Organization of the Indian hedgehog–parathyroid hormone-related protein system in the postnatal growth plate. J Mol Endocrinol. 2011;47(1):99-107.

[2] Retting KN, Song B, Yoon BS,et al. BMP canonical Smad signaling through Smad1 and Smad5 is required for endochondral bone formation. Development. 2009;136(7): 1093-1104.

[3] Johnson RW, Nguyen MP, Padalecki SS,et al. TGF-beta promotion of Gli2-induced expression of parathyroid hormone-related protein, an important osteolytic factor in bone metastasis, is independent of canonical Hedgehog signaling. Cancer Res. 2011; 71(3):822-831.

[4] Amano K,Hata K,Sugita A,et al.Sox9 family members negatively regulate maturation and calcification of chondrocytes through up-regulation of parathyroid hormone-related protein. Mol Biol Cell. 2009; 20(21): 4541-4551.

[5] Kim SY,Im GI.The expressions of the SOX trio, PTHrP (parathyroid hormone-related peptide)/IHH (Indian hedgehog protein) in surgically induced osteoarthritis of the rat. Cell Biol Int. 2011; 35(5):529-535.

[6] Antonio Casado-Díaz, Raquel Santiago-Mora, José Manuel Quesada.The N- and C-terminal domains of parathyroid hormone-related protein affect differently the osteogenic and adipogenic potential of human mesenchymal stem cells. Exp Mol Med. 2010;42(2):87-98.

[7] Strid H,Care A,Jansson T, Powell T. Parathyroid hormone-related peptide (38-94) amide stimulates ATP-dependent calcium transport in the Basal plasma membrane of the human syncytiotrophoblast. J Endocrinol. 2002;175(2): 517-524.

[8] Alonso V, de Gortazar AR, Ardura JA, et al. Parathyroid hormone-related protein (107-139) increases human osteoblastic cell survival by activation of vascular endothelial growth factor receptor-2. J Cell Physiol.2008;217: 717-727.

[9] ZHANG Shu-wei, CHEN An-min, LI Ming-hui,et al. Establishment of pTet-on rat precartilaginous stem cells and construction of pTRE-PTHrP(1-36) responsive plasmids. Chin J Ttauma.2008;24(7):564-568.

[10] Wübbenhorst D,Dumler K,Wagner B,et al. Tetracycline- regulated bone morphogenetic protein 2 gene expression in lentivirally transduced primary rabbit chondrocytes for treatment of cartilage defects. Arthritis Rheum. 2010;62(7): 2037-2046.

[11] Zuyi Huang, Colby Moya, Arul Jayaraman, et al. Using the Tet-On system to develop a procedure for extracting transcription factor activation dynamics. Mol. BioSyst. 2010; 10(6):1883-1889.

[12] Kouta Mayanagia, Shinichi Kiyonarib, Hirokazu Nishidac,et al. Architecture of the DNA polymerase B-proliferating cell nuclear antigen (PCNA)-DNA ternary complex. PNAS.2011; 108(5):1845-1849.

[13] Huang CY, Lai KY, Hung LF, et al. Advanced glycation end products cause collagen II reduction by activating Janus kinase/signal transducer and activator of transcription 3 pathway in porcine chondrocytes. Rheumatology (Oxford). 2011;50(8):1379-1389.

[14] Petit A, Demers CN, Girard-Lauriault PL, et al. Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes. Biomed Eng Online. 2011;10:4.

[15] Levi B, James AW, Nelson ER,et al. Role of Indian Hedgehog Signaling in Palatal Osteogenesis. Plastic & Reconstructive Surgery.2011; 127(3):1182-1190.