Chloride-hydrogen antiporters ClC-3 and ClC-5 drive osteoblast mineralization and regulate fine-structure bone patterning in vitro

Abstract

Osteoblasts form an epithelium-like layer with tight junctions separating bone matrix from extracellular fluid. During mineral deposition, calcium and phosphate precipitation in hydroxyapatite liberates 0.8 mole of H+ per mole Ca+2. Thus, acid export is needed for mineral formation. We examined ion transport supporting osteoblast vectorial mineral deposition. Previously we established that Na/H exchangers 1 and 6 are highly expressed at secretory osteoblast basolateral surfaces and neutralize massive acid loads. The Na/H exchanger regulatory factor-1 (NHERF1), a pdz-organizing protein, occurs at mineralizing osteoblast basolateral surfaces. We hypothesized that high-capacity proton transport from matrix into osteoblast cytosol must exist to support acid transcytosis for mineral deposition. Gene screening in mineralizing osteoblasts showed dramatic expression of chloride–proton antiporters ClC-3 and ClC-5. Antibody localization showed that ClC-3 and ClC-5 occur at the apical secretory surface facing the bone matrix and in membranes of buried osteocytes. Surprisingly, the Clcn3−/− mouse has only mildly disordered mineralization. However, Clcn3−/− osteoblasts have large compensatory increases in ClC-5 expression. Clcn3−/− osteoblasts mineralize in vitro in a striking and novel trabecular pattern; wild-type osteoblasts form bone nodules. In mesenchymal stem cells from Clcn3−/− mice, lentiviral ClC-5 shRNA created Clcn3−/−, ClC-5 knockdown cells, validated by western blot and PCR. Osteoblasts from these cells produced no mineral under conditions where wild-type or Clcn3−/− cells mineralize well. We conclude that regulated acid export, mediated by chloride–proton exchange, is essential to drive normal bone mineralization, and that CLC transporters also regulate fine patterning of bone.

Osteoblast Differentiation and Bone Matrix FormationIn VivoandIn Vitro

“…This process allows mineral formation to proceed to completion at varying serum calcium and phosphate. 5 Once deposited, the mineral undergoes maturation from amorphous calcium phosphate to hydroxyapatite, with important changes in mineral orientation and organization. This surprisingly slow and complex process depends critically on pH.…”

“…2C). 5 This process and osteoblast phosphate transport are regulated by the sodium-hydrogen exchanger regulatory factor-1. 22 It is established that osteoblast apical membrane phosphatases, particularly alkaline phosphatase, also to some extent PHOSPHO1, and in a complex and sometimes inhibitory manner ENPP1, 55,56 regulate phosphate production that is essential for mineralization.…”

“…2D). 5 The phosphate supply is normal, but the osteoblasts cannot make the gradient that drives mineral transport to completion (Equation 1). Important processes are not accounted by this model: Osteoid is a dense collagen hydrogel; when osteoid is mineralized, the water is eliminated.…”

Adrenocorticotropic hormone and 1,25-dihydroxyvitamin D3 enhance human osteogenesis in vitro by synergistically accelerating the expression of bone-specific genes

“…This reflects that bone mineral is not a result of mass action; hydroxyapatite precipitation occurs in a closed environment and is driven to completion by outward transport of the acid produced by mineral precipitation. 22,34 This physiology is the ‘Achilles heel’ in translating in vitro effects on osteoblast differentiation into clinical results; the interesting effects of 1,25(OH) 2 D on ACTH action and, in particular, VEGF production are more likely to be physiologically important. 1 Particularly, the strong and reproducible increase in osteoblast mineral production in vitro in high calcium does not translate to bone production in response to oral calcium in the setting of adequate calcium intake.…”

Long-read assembly of the Chinese rhesus macaque genome and identification of ape-specific structural variants

“…whole-genome shotgun, BAC: bacterial artificial chromosome; *only N-base regions in the assembled chromosomes were counted; †only chromosome-placed bases were counted; ‡non-N bases (bp) in the assembly. (De Nys, et al 2001;Kondo, et al 2017;Larrouture, et al 2015;Moon and Parker 2018;Perez-Duran, et al 2012;Riazanski, et al 2011;Shin, et al 2017;Siep, et al 2004 The chromosomal distribution of contigs of the rheMacS genome assembly. We compare genome sequence contiguity between rheMac8 and rheMacS.…”