Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density
Fig 6
An in silico bone turnover model validated class 2 candidate BMD genes.
(A) The in silico model of bone turnover captured the extracellular break down of bone and the subsequent formation of new bone. The model is simplified excluding for example many of the intracellular proteins driving bone turnover or non-collagenous matrix proteins. While resorption of both bone matrix and mineral may occur concurrent, bone matrix formation typically precedes mineral deposition. The genes mainly responsible for bone turnover were grouped and annotated as follows. Osteoclastic bone matrix resorption: tartrate-resistant acid phosphatase type 5 (Acp5), essential for osteoclast motility; cathepsin K (Ctsk), matrix break down; matrix metallopeptidase 9, (Mmp9), matrix break down; matrix metallopeptidase 14 (Mmp14), matrix break down. Osteoclastic bone mineral resorption: ATPase H+ transporting V0 subunit D2 (Atp6v0d2), acidic pH and mineral removal; ATPase H+ transporting V0 subunit A3, (Tcirg1), acidic pH and mineral removal. Osteoblastic bone matrix formation: ADAM metallopeptidase with thrombospondin type 1 motif 2. (Adamts2), N-terminal processing of procollagen; ADAM metallopeptidase with thrombospondin type 1 motif 14 (Adamts14), N-terminal processing of procollagen; bone morphogenic protein 1 (Bmp1), C-terminal processing of procollagen; collagen type I alpha 1 chain (Col1a1) and collagen type I alpha 2 chain (Col1a2), procollagen formation; procollagen C-endopeptidase enhancer 1 (Pcolce), enhancer of C-terminal processing of procollagen; tolloid like 1 (Tll1), C-terminal processing of procollagen. Osteoblastic bone mineral deposition: tissue-nonspecific alkaline phosphatase (Alpl), removal of mineralization inhibitor inorganic pyrophosphate (PPi); ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1), production of PPi. Together, these proteins (red elliptic nodes) formed the PPI networks for osteoclastic resorption and osteoblastic formation during bone turnover. Connecting proteins distinct from the bone turnover genes are represented as pink elliptic nodes, while white nodes marked genes with an undefined role in bone. Black lines represent PPIs as determined by STRING or REACTOME analysis. Arrows indicated REACTOME-curated data. For validation, we used experimental gene expression data from osteoclasts and osteoblasts in combination with statistical testing. The resulting gaussian probability of interactions, i.e. FDR-adjusted p-values (q-values) were superimposed on the STRING PPIs as follows: large line width, q<0.0005; medium line width, q<0.05; small line width, q>0.05 or no q data. (B) Based on their cellular expression, the three class 2 low BMD candidate genes Rab31p, Ncald, and Arl4d were probed in the osteoclastic and osteoblastic compartments of bone turnover. Rab3ip and Ncald interacted via single intermediates with proteins degrading bone matrix and also mineral, while Arl4d, also via an intermediate, interacted with a regulator of the procollagen cleavage essential for bone matrix formation.