Strain Reduction of Human Gingival Fibroblasts Induces the ATP Pathway

Abstract

Studies in rats demonstrated that surgical detachment of marginal gingiva from root surfaces induced alveolar bone resorption via activation of ATP receptor P2X4. Our aim was to study the effects of strain release of human gingival fibroblasts by detachment of collagen coat from culture dishes. Changes in cell shape, extracellular ATP, cell calcium and genes relevant to trigger alveolar bone resorption were measured. HGF cells from human marginal gingiva explants were seeded on collagen coated culture dishes. In the test group, the collagen coat was detached from culture dish 4-5 days after seeding. Quantification of extracellular ATP was measured by Bioluminescent Assay Kit and changes of intracellular calcium by FLU-AM fluorescence. Real-time PCR was used to examine expression of purinergic receptor P2X4 and P2X7, RANK-L and STC1. Cell length of HGFs grown on plastic surface and on collagen was similar. Reduction in cell length of 45% was measured after detachment of collagen coat. Extracellular ATP raised 10 folds 1 minute after detachment of collagen coat, then declined and returned to control levels 60 minutes later. Ionomycin increased extracellular ATP, while, pretreatment by EGTA, or BAPTA-AM, 30 minutes prior to Ionomycin, reduced significantly extracellular ATP. Reciprocally, addition of ATP increased influx of calcium into HGFs. At the molecular level elevated expression of STC-1, RANK-L and P2X7 was recorded by RT -PCR in detached HGFs, while expression of P2X4 was unchanged. Expression of STC-1, a modulator of cellular calcium and phosphate, P2X7 a calcium ionic channel purinergic receptor and RANK-L a powerful regulator of osteoclastic activity, all were up-regulated significantly. Conclusion:We propose that drop of strain of HGF cells and change in their shape stimulated release of cellular ATP which is signaling through Pi/Ca modulators the molecular activation of osteoclasts.

Periodontal cell mechanotransduction

“…The mechanical properties of cells may play an important role in the progression and establishment of PD. Yaffe and co-workers proposed the strain relaxation hypothesis for explaining alveolar bone resorption in PD nearly 15 years ago [103], and have continued to argue for it in a series of more recent papers [107,108]. They proposed that relaxation of tension in collagen bundles due to collagen degradation in the gingiva stimulates alveolar bone resorption [103].…”

Role of Mechanotransduction in Periodontal Homeostasis and Disease

“…It has been described that PDLFs show altered shapes in response to mechanical stimulation, which may result from decreased outside-in actomyosin and consequently nuclear tension. The altered cell shapes upregulate exogenous ATP levels and ATP receptor expression with RANK-L (receptor activator of nuclear factor kappa-B ligand), all of which stimulate alveolar bone resorption, offering another insight into the ECM-cytosol-nucleus signaling network in the periodontal context (Gadban et al 2015).…”

From the Matrix to the Nucleus and Back: Mechanobiology in the Light of Health, Pathologies, and Regeneration of Oral Periodontal Tissues

“…The ATP-associated signaling cascades result in an increase in the receptor activator of nuclear factor kappa-B ligand (RANK-L) expression in PDLFs, which contributes to alveolar bone resorption. The latter process, which is important for periodontal regeneration disease, might, therefore, be in part the macroscopic correlate of periodontal NMT [220].…”