Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone

Abstract

Extracellular matrix (ECM) mineralization is a physiological process in bone and a pathological one in soft tissues. The mechanisms determining the spatial restriction of ECM mineralization to bone physiologically are poorly understood. Here we show that a normal extracellular phosphate concentration is required for bone mineralization, while lowering this concentration prevents mineralization of any ECM. However, simply raising extracellular phosphate concentration is not sufficient to induce pathological mineralization, this is because of the presence in all ECMs of pyrophosphate, an inhibitor of mineralization. ECM mineralization occurs only in bone because of the exclusive coexpression in osteoblasts of Type I collagen and Tnap, an enzyme that cleaves pyrophosphate. This dual requirement explains why Tnap ectopic expression in cells producing fibrillar collagen is sufficient to induce pathological mineralization. This study reveals that coexpression in osteoblasts of otherwise broadly expressed genes is necessary and sufficient to induce bone mineralization and provides evidence that pathological mineralization can be prevented by modulating extracellular phosphate concentration.

Figure 1.

Role of mineral ions in inducing ECM mineralization. (A–C) Extracellular mineral deposition as a function of P_i concentration. Wild-type (WT) mouse osteoblasts were cultured in the presence of either constant extracellular Ca and increasing amount of P_i (Na₂HPO₄), or constant P_i and increasing amount of calcium (CaCl₂). As shown by von Kossa and Alizarin red staining, significant ECM mineralization occurred with increasing P_i concentration. (D) In vitro mineralization of wild-type (WT) and Hyp osteoblasts. In the presence of 5 mM P_i in culture media, ECM surrounding Hyp osteoblasts mineralized at the same pace and to the same extent as wild-type osteoblasts. (E) Von Kossa and van Gieson staining of vertebrae from 1-mo-old wild-type (WT) mice fed a normal diet and Hyp mice fed a normal or a high-phosphorus diet. Feeding of a high-phosphorus diet nearly normalized bone mineralization in Hyp mice (n = 6).

Figure 2.

Extracellular P_i and ectopic ECM mineralization. (A) Alizarin red-stained skeletal preparation (thorax) of 1-mo-old wild-type (WT), Mgp^–/– and Mgp^–/–; Hyp mice. The arrow indicates mineralized aorta in Mgp^–/– mice. Note that unlike the Mgp^–/– aorta, the aorta of a Mgp^–/–; Hyp mouse did not mineralize. (B) Mgp^–/–; Hyp mice had a normal life span (n = 10). (C) Von Kossa staining of Mgp^–/–; Hyp aorta sections showed no mineral deposition. Note extensive mineral deposition in arteries of 3-mo-old Mgp^–/–; Hyp mice when fed a high-phosphorus diet. (D) X-rays showing ectopic mineral deposition in joints of 4-mo-old ank mouse (arrow) but not in ank; Hyp mouse. (E) ank; Hyp mice had a normal life span (n = 8). (F) Von Kossa and van Gieson staining of 3-mo-old wild-type (WT); ank and ank; Hyp joints. ank; Hyp mice fed a normal diet had no articular erosion. When fed a high-phosphorus diet ank; Hyp mice developed extensive joint mineralization.

Figure 3.

Pyrophosphate as a physiological inhibitor of ECM mineralization. (A) Northern blot. Ubiquitous expression of ank and Enpp1. (B) Increasing serum P_i level enhanced joint mineralization in 6-wk-old ank and Enpp1^–/– mice (n = 3). (C) Increased extracellular P_i resulted in arterial ECM mineralization in 6-wk-old ank and Enpp1^–/– mice fed a high-phosphorus diet but not when fed a normal diet (n = 3). (D) ECM mineralization of dermis in 6-wk-old Enpp1^–/– mice fed a high-phosphorus diet but not when fed a normal diet. (E) Transmission electron microscopy (TEM) showing deposition of mineral crystals along the collagen fibrils. (F) Electron diffraction confirmed that the deposited mineral was hydroxyapatite.

Figure 4.

TNAP function and ECM mineralization. (A) Wild-type (WT) osteoblasts staining with Fast Blue confirming TNAP activity. ECM when culture media were supplemented either by β-glycerophosphate (βGP) or PP_i. Tnap^–/– osteoblasts did not stain blue and did not mineralize in either condition. When culture media were supplemented by 5 mM P _i, Tnap^–/– ECM surrounding osteoblasts also mineralized albeit to a lesser extent than wild-type (WT) osteoblasts. (B, top) Transgene construct for liver-specific expression of Tnap. (Bottom) Northern analysis using a 3′ probe specific for human Tnap cDNA showed liver-specific expression of the transgene. (C) TNAP produced by the transgene was biologically active as it released phosphate from βGP at a higher rate than the wild-type (WT) serum. (D) Increased TNAP serum concentration in ApoE-Tnap; Tnap^–/– mice prevented hyperosteoidosis caused by TNAP deficiency in Tnap^–/– mice (n = 3).

Figure 5.

Type I collagen scaffold and ECM mineralization. (A) Northern blot showing coexpression of Tnap, Col1a1, and Col1a2 only in skeletal tissues. (B) Northern blot analysis showing expression of osteoblast-specific genes in ROS 17/2.8 cells, primary osteoblasts (Ob), and Atf4^–/– cells. Expression of Col1a1 is markedly down-regulated in ROS 17/2.8 cells. (C, top) Von Kossa staining showing a reduced mineralization of the ECM surrounding ROS 17/2.8 and Atf4^–/– osteoblasts cultured in the presence of β-glycerophosphate (βGP) in comparison to wild-type (WT) osteoblasts. (Bottom) Van Gieson staining showed ROS 17/2.8 and Atf4^–/– osteoblasts synthesized less collagen. ROS 17/2.8 cells transfected with a Col2a1 expression vector produced a collagenous matrix (van Gieson staining) and mineralized when cultured in presence of βGP. No ECM mineralization was seen in the case of empty vector transfected ROS cells.

Figure 6.

Coexpression of Tnap and Type I collagen is required for ECM mineralization. (A) NIH3T3 cells transfected with a Tnap expression vector produced TNAP as shown by Fast Blue staining. ECM surrounding these cells mineralized when cultured in presence of β-glycerophosphate (βGP) or PP_i. No ECM mineralization was seen in the case of empty vector transfected NIH3T3 cells. (B) Van Gieson staining (pink) of skin showing collagen in the dermis but not in the epidermis. (C, top) Transgene constructs for dermis- and epidermis-specific expression of Tnap. (Bottom) Fast Blue staining (blue) showing TNAP activity in the epidermis of K14-Tnap mice and dermis of α2(I)-Tnap mice. (D) Von Kossa staining showing massive mineral deposition in dermis of α2(I)-Tnap mouse, while no mineral deposition was seen in the epidermis of K14-Tnap mouse (n = 6). (E, middle) Micro-CT analysis of the mineralized tail of a α2(I)-Tnap mouse. Similar analysis with wild type (WT) (left) and K14-Tnap (right) presented for comparison. (F) Electron micrograph showing mineral deposition along collagen fibrils in the α2(I)-Tnap mouse.