Inhibition of PHOSPHO1 activity results in impaired skeletal mineralization during limb development of the chick

Abstract

PHOSPHO1 is a bone-specific phosphatase implicated in the initiation of inorganic phosphate generation for matrix mineralization. The control of mineralization is attributed to the actions of tissue-nonspecific alkaline phosphatase (TNAP). However, matrix vesicles (MVs) containing apatite crystals are present in patients with hypophosphatasia as well as TNAP null (Akp2(-/-)) mice. It is therefore likely that other phosphatases work with TNAP to regulate matrix mineralization. Although PHOSPHO1 and TNAP expression is associated with MVs, it is not known if PHOSPHO1 and TNAP are coexpressed during the early stages of limb development. Furthermore, the functional in vivo role of PHOSPHO1 in matrix mineralization has yet to be established. Here, we studied the temporal expression and functional role of PHOSPHO1 within chick limb bud mesenchymal micromass cultures and also in wild-type and talpid(3) chick mutants. These mutants are characterized by defective hedgehog signalling and the absence of endochondral mineralization. The ability of in vitro micromass cultures to differentiate and mineralize their matrix was temporally associated with increased expression of PHOSPHO1 and TNAP. Comparable changes in expression were noted in developing embryonic legs (developmental stages 23-36HH). Micromass cultures treated with lansoprazole, a small-molecule inhibitor of PHOSPHO1 activity, or FGF2, an inhibitor of chondrocyte differentiation, resulted in reduced alizarin red staining (P<0.05). FGF2 treatment also caused a reduction in PHOSPHO1 (P<0.001) and TNAP (P<0.001) expression. Expression analysis by whole-mount RNA in situ hybridization correlated with qPCR micromass data and demonstrated the existence of a tightly regulated pattern of Phospho1 and Tnap expression which precedes mineralization. Treatment of developing embryos for 5 days with lansoprazole completely inhibited mineralization of all leg and wing long bones as assessed by alcian blue/alizarin red staining. Furthermore, long bones of the talpid(3) chick mutant did not express Phospho1 or Tnap whereas flat bones mineralized normally and expressed both phosphatases. In conclusion, this study has disclosed that PHOSPHO1 expression mirrors that of TNAP during embryonic bone development and that PHOSPHO1 contributes to bone mineralization in developing chick long bones.

Keywords: Alkaline Phosphatase; PHOSPHO1; chondrocyte differentiation; mineralization; talpid3.

Fig. 1

Quantification of (A) alcian blue and (B) alizarin red staining in micromasses derived from stage 23HH limb bud mesenchymal cells, which were cultured for 0, 3, 7 and 10-days. Data are presented as the mean +/− S.E.M. of six observations within each time point. ** P<0.01; ***P<0.001. Expression of Collagen type II by PCR in (C) micromass cultures maintained for up to 10-days in culture and (D) chick limb tissue at stages 23-36HH.

Fig. 2

Fold changes in the mRNA expression of (A, D) Phospho1 and (B, E) Tnap in (A, B) micromasses prepared from stage 23HH limb bud mesenchymal cells and cultured for 0, 3, 7 and 10-days or in (D, E) chick limb tissue at stages 23-36HH. PHOSPHO1 and TNAP protein expression in corresponding (C) micromasses cultured for 0-10d and (F) tissues at 23-36HH. Data are presented as the mean +/− S.E.M. of six observations within each culture time point or of 4 limb buds/limbs of each developmental age. * P<0.05; **P<0.01; ***P<0.001.

Fig. 3

Whole-mount RNA in-situ hybridization showing Tnap and Phospho1 expression during development. (A) Expression of Phospho1 at stage 23HH of development. (B) Expression of Tnap at stage 23HH of development. (C) Expression of Phospho1 in the leg at stage 26HH of development. (D) Expression of Tnap in the leg at stage 26HH of development. (E) Contralateral wings showing expression of Phospho1 (left) and Tnap (right) at stage 31HH, arrows indicates metacarpal of digit 3. (F) Contralateral legs showing expression of Phospho1 (left) and Tnap (right) at stage 31HH, arrowheads indicate expression in the metatarsals. (G) Contralateral wings showing expression of Phospho1 (left) and Tnap (right) at stage 35HH. (H) Contralateral legs showing expression of Phospho1 (left) and Tnap (right) at stage 35HH, arrow indicates Tnap expression in phalanx1 of digit 3. Abbreviations: R=radius, U=ulna, MT=metatarsals.

Fig. 4

Quantification of (A) alcian blue (B) alizarin red and (C) sirius red staining in 7-day cultured micromasses derived from stage 20 limb bud mesenchymal cells in the presence of FGF2 (50ng/ml). Fold changes in the mRNA expression of (D) Phospho1 and (E) Tnap in 7-day cultured micromasses derived from stage 20 limb bud mesenchymal cells in the presence of FGF2 (50ng/ml). Data are presented as the mean +/− S.E.M. of six observations within each treatment. * P<0.05; ***P<0.001 (F) PHOSPHO1 and TNAP protein expression in corresponding 7-day cultured micromasses.

Fig. 5

(A) Expression of Collagen type X by PCR in 3 individual control and 7-day lansoprazole treated micromass cultures maintained for up to 10-days in culture. (B) Effect of 3-day and 7-day lansoprazole exposure (100μM) on micromass alizarin red staining. Data are presented as the mean +/− S.E.M. of six observations within each treatment. NS: not significant. * P<0.05; **P<0.01

Fig: 6

Whole-mount alcian blue/alizarin red staining showing cartilaginous matrix/mineral at 10-days of development following exposure to DMSO (A, C) or lansoprazole (B, D) in the long bones of the wing and leg. In comparison to the mineralization centers of the control limbs (A, C; asterisks) little or no mineralization was noted in the diaphysis of the long bones of lansoprazole treated embryos (B, D; asterisks).

Fig. 7

Whole-mount alcian blue/alizarin red staining of wild-type (A, C) and talpid³ (B, D) embryos at 10-days of development, clavicles are marked by an asterisk, mineralization of the humerus and femur of wild-type embryos and equivalent regions in talpid³ embryos indicated by arrows. Although the long bones of the mutant talpid³ embryos are poorly formed individual long bones are still clearly seen not to be mineralized which is in contrast to their wild-type counterparts. The clavicle is mineralized in both the wild-type and talpid³ embryos. (E-H) Expression of Tnap in wild-type wing (E; asterisk denotes clavicle), femur (G) and talpid³ wing (F, asterisk denotes clavicle) and femur (H). Figs. I-L. Expression of Phospho1 in wild-type wing (I; asterisk denotes clavicle), femur (K) and talpid³ wing (J, asterisk denotes clavicle) and femur (L).