cDNA Cloning of S100 Calcium-binding Proteins from Bovine Periodontal Ligament and Their Expression in Oral Tissues

Abstract

The periodontal ligament (PDL) is a unique tissue that is crucial for tooth function. However, little is known of the molecular mechanisms controlling PDL function. To characterize PDL cells at the molecular level, we constructed a cDNA library from bovine PDL tissue. We then focused on the isolation of S100 calcium-binding proteins (CaBPs), because they mediate Ca²⁺ signaling and control important cellular processes such as differentiation and metabolism. We screened the PDL cDNA library with a mouse S100A4 cDNA, and cloned the bovine cDNAs of two S100 CaBPs (S100A4 and S100A2). In Northern blotting analysis, the highest expression of S100A4 was detected in PDL from erupted teeth (PDLE). PDL from teeth under eruption (PDLU) showed a lower expression of S100A4, and its expression in gingiva was faintly detectable. S100A4 expression was also high in the pulp tissue followed by the dental papilla of the tooth germ. S100A2 expression was high in PDLE and gingiva. Interestingly, only PDLE exhibited a high expression of both S100A4 and S100A2. PDLE also expressed the highest level of β-actin, a target cytoskeletal protein for S100A4. It is conceivable that the high expression of S100A4 in PDLE is a result of the maturation of the PDL and/or a response to mechanical stress generated by mastication. Since there was a marked difference of S100A4 expression between PDL and gingiva, we propose that S100A4 could be a useful marker for distinguishing cells from these two tissues.

Keywords

S100 calcium-binding proteins periodontal ligament oral tissues cDNA cloning.

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References

Arceo N. , Sauk JJ , Moehring J. , Foster RA , Somerman MJ (1991). Human periodontal cells initiate mineral-like nodules in vitro. J Periodontol 62:499-503.

Biragyn A. , Arkins S. , Kelley KW (1994). Riboprobe expression cassettes for measuring IGF-I, beta-actin and glyceraldehyde 3-phosphate dehydrogenase transcripts. J Immunol Meth 168:235-244.

Carlyle WC , Toher CA , Vandervelde JR , McDonald KM , Homans DC , Cohn JN ( 1996). Changes in beta-actin mRNA expression in remodeling canine myocardium. J Mol Cell Cardiol 28:53-63.

Chomczynski P. , Sacchi N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 162:156-159.

Davidson RM , Tatakis DW , Auerbach AL (1990). Multiple forms of mechanosensitive ion channels in osteoblast-like cells. Pflügers Arch 416:646-651.

Davidson RM (1993). Membrane stretch activates a high-conductance K+ channel in G292 osteoblastic-like cells. J Membrane Biol 131:81-92.

Giannopoulou C. , Cimasoni G. (1996). Functional characteristics of gingival and periodontal ligament fibroblasts. J Dent Res 75:895-902.

Glenney JR Jr , Kindy MS , Zokas L. (1989). Isolation of a new member of the S100 protein family: amino acid sequence, tissue, and subcellular distribution. J Cell Biol 108:569-578.

Glogauer M. , Ferrier J. , McCulloch CA (1995). Magnetic fields applied to collagen-coated ferric oxide beads induce stretch-activated Ca2+ flux in fibroblasts. Am J Physiol 269:C1093-C1104.

10.

Glogauer M. , Arora P. , Yao G. , Sokholov I. , Ferrier J. , McCulloch CA (1997). Calcium ions and tyrosine phosphorylation interact coordinately with actin to regulate cytoprotective responses to stretching . J Cell Sci 110:11-21.

11.

Goto K. , Endo H. , Fujiyoshi T. (1987). Cloning of the sequences expressed abundantly in established cell lines: identification of a cDNA clone highly homologous to S-100, a calcium binding protein. J Biochem 103:48-53.

12.

Gunning P. , Ponte P. , Okayama H. , Engel J. , Blau H. , Kedes L. (1983). Isolation and characterization of full-length cDNA clones for human a-, β-, and T-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed. Mol Cell Biol 3:787-795.

13.

Kriajevska MV , Cardenas MN , Grigorian MS , Ambartsumian NS , Georgiev GP , Lukanidin EM (1994). Non-muscle myosin heavy chain as a possible target for protein encoded by metastasis-related mts-1 gene. J Biol Chem 269:19679-19682.

14.

Moxham BJ , Grant DA (1995). Development of the periodontal ligament. In: The periodontal ligament in health and disease. Berkovitz BKB , Moxham BJ , Newman HN , editors. Barcelona: Mosby-Wolfe, pp. 161-181.

15.

Mukai M. , Yoshimine Y. , Akamine A. , Maeda K. (1993). Bone-like nodules formed in vitro by rat periodontal ligament cells. Cell Tissue Res 271:453-460.

16.

Nagahara S. , Matsuda T. (1996). Cell-substrate and cell-cell interactions differently regulate cytoskeletal and extracellular matrix protein gene expression. J Biomed Mater Res 32:677-686.

17.

Nakago-Matsuo C. , Matsuo T. , Nakago T. (1996). Intracellular calcium response to hydraulic pressure in human periodontal ligament fibroblasts. Am J Orthod Dentofac Orthop 109:244-248.

18.

Nohutcu RM , Somerman MJ , McCauley LK (1995). Dexamethasone enhances the effects of parathyroid hormone on human periodontal ligament cells in vitro. Calcif Tissue Int 56:571-577.

19.

Nohutcu RM , McCauley LK , Shigeyama Y. , Somerman MJ (1996). Expression of mineral-associated proteins by periodontal ligament cells: in vitro vs. ex vivo. J Periodont Res 31:369-372.

20.

Pedrocchi M. , Schafer BW , Mueller H. , Eppenberger U. , Heizmann CW (1994). Expression of Ca2+-binding proteins of the S100 family in malignant human breast-cancer cell lines and biopsy samples. Int J Cancer 57:684-690.

21.

Pender N. , McCulloch CA (1991). Quantitation of actin polymerization in two human fibroblast sub-types responding to mechanical stretching. J Cell Sci 100:187-193.

22.

Polans AS , Palczewski K. , Asson-Batres MA , Ohguro H. , Witkowska D. , Haley TL , et al. (1994). Purification and primary structure of Capl, an S-100-related calcium-binding protein isolated from bovine retina. J Biol Chem 269:6233-6240.

23.

Ramakrishnan PR , Lin WL , Sodek J. , Cho MI (1995). Synthesis of noncollagenous extracellular matrix proteins during development of mineralized nodules by rat periodontal ligament cells in vitro. Calcif Tissue Int 57:52-59.

24.

Sanger F. , Nicklen S. , Coulson AR (1977). DNA sequencing with chain-terminating inhibitors . Proc Natl Acad Sci USA 74:5463-5467.

25.

Schafer BW , Heizmann CW (1996). The S100 family of EF-hand calcium-binding proteins: functions and pathology (review). Trends Biochem Sci 21:134-140.

26.

Spanakis E. (1993). Problems related to the interpretation of autoradiographic data on gene expression using common constitutive transcripts as controls . Nucleic Acids Res 21:3809-3819.

27.

Strutz F. , Okada H. , Lo CW , Danoff T. , Carone RL , Tomaszewski JE , et al. (1995). Identification and characterization of a fibroblast marker: FSP1. J Cell Biol 130:393-405.

28.

Takenaga K. , Nakamura Y. , Sakiyama S. , Hasegawa Y. , Sato K. , Endo H. ( 1994). Binding of pEL98 protein, an S100-related calcium-binding protein, to nonmuscle tropomyosin. J Cell Biol 124:757-768.

29.

Wang N. , Butler JP , Ingber DE (1993). Mechanotransduction across the cell surface and through the cytoskeleton. Science 260:1124-1127.

30.

Watanabe Y. , Usada N. , Minami H. , Morita T. , Tsugane S. , Ishikawa R. , et al. (1993). Calvasculin, as a factor affecting the microfilament assemblies in rat fibroblasts transfected by src gene. FEBS Lett 324:51-55.