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Cyclic Mechanical Strain Regulates the PTHrP Expression in Cultured Chondrocytes via Activation of the Ca²⁺ Channel

N. Tanaka^1,, S. Ohno^1,*,, K. Honda¹, K. Tanimoto¹, T. Doi¹, M. Ohno-Nakahara¹, E. Tafolla², S. Kapila³, and K. Tanne¹

¹ Departments of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-Ku, Hiroshima 734-8553, Japan;
² Department of Stomatology, University of California-San Francisco, San Francisco, CA, USA; and
³ Department of Orthodontics and Pediatric Dentistry, School of Dentistry, University of Michigan, Ann Arbor, USA;

View larger version (50K): [in a new window]   Figure 1. Effect of mechanical strain on chondrocyte metabolism. (A) The cell appearance in the cultures with (b,d) or without (a,c) 24-hour mechanical strain of 12% elongation at day 4 (a,b) and day 12 (c,d). Bar = 3 µm. The magnified appearance of a dividing cell is shown in the inset. The syntheses of DNA per well for proliferating chondrocytes (on day 4) (B), proteoglycan (C), and collagen (D) per well for matrix-forming chondrocytes (on day 12) were determined as described in MATERIALS & METHODS. = negative stress; = positive stress. Mean ± SD, n = 3, *p < 0.05.

View larger version (20K): [in a new window]   Figure 2. Effect of mechanical strain on PTHrP expression in chondrocytes. (A) The expression of PTHrP mRNA varies during chondrocyte differentiation in cultured growth plate chondrocytes. The time-dependent expressions of PTHrP mRNA were determined at four-day intervals between days 4 and 24 of culture by real-time RT-PCR. Values were normalized with the expression level of GAPDH and denoted as the fold-increase of PTHrP mRNA expression relative to those of day 4 (proliferating) chondrocytes. (B) Cyclic mechanical strain induces PTHrP mRNA that is dependent upon the status of cellular differentiation. Proliferating, matrix-forming, and hypertrophic chondrocytes were subjected to cyclic mechanical strains of 7% or 12% elongation for 12 hrs, and the expression of PTHrP mRNA was examined by real-time RT-PCR. Values were normalized with the expression level of GAPDH and denoted as the fold-increase of PTHrP mRNA expression relative to control samples (0% elongation). Mean ± SD, n = 3, **p < 0.01, *p < 0.05.

View larger version (42K): [in a new window]   Figure 3. Ca2+ channel blocker, but not the SA or Na+ or K+ channel blocker, inhibits the mechanical induction of PTHrP mRNA. Day 12 (matrix-forming) chondrocytes were subjected to a cyclic mechanical strain of 12% elongation in the presence of different channel blockers for 12 hrs, and the expression of PTHrP mRNA was examined by real-time RT-PCR. Values were normalized with the expression level of GAPDH and denoted as the fold-increase of PTHrP mRNA expression relative to control samples (0% elongation; without channel blockers). Mean ± SD, n = 3, **p < 0.01, *p < 0.05.