In Vivo Overexpression of Tissue-Nonspecific Alkaline Phosphatase Increases Skeletal Mineralization and Affects the Phosphorylation Status of Osteopontin

Abstract

Functional ablation of tissue-nonspecific alkaline phosphatase (TNAP) (Alpl^−/− mice) leads to hypophosphatasia, characterized by rickets/osteomalacia attributable to elevated levels of extracellular inorganic pyrophosphate, a potent mineralization inhibitor. Osteopontin (OPN) is also elevated in the plasma and skeleton of Alpl^−/− mice. Phosphorylated OPN is known to inhibit mineralization, however, the phosphorylation status of the increased OPN found in Alpl^−/− mice is unknown. Here, we generated a transgenic mouse line expressing human TNAP under control of an osteoblast-specific Col1a1 promoter (Col1a1-Tnap). The transgene is expressed in osteoblasts, periosteum, and cortical bones, and plasma levels of TNAP in mice expressing Col1a1-Tnap are 10-20 times higher than those of wild-type mice. The Col1a1-Tnap animals are healthy and exhibit increased bone mineralization by microCT analysis. Crossbreeding of Col1a1-Tnap transgenic mice to Alpl^−/− mice rescues the lethal hypophosphatasia phenotype characteristic of this disease model. Osteoblasts from [Col1a1-Tnap] mice mineralize better than non-transgenic controls and osteoblasts from [Col1a1-Tnap^+/−; Alpl^−/−] mice are able to mineralize to the level of Alpl^+/− heterozygous osteoblasts, while Alpl^−/− osteoblasts show no mineralization. We found that the increased levels of OPN in bone tissue of Alpl^−/− mice are comprised of phosphorylated forms of OPN while WT and [Col1a1-Tnap^+/−; Alpl^−/−] mice had both phosphorylated and dephosphorylated forms of OPN. OPN from [Col1a1-Tnap] osteoblasts were more phosphorylated than non-transgenic control cells. Titanium dioxide-liquid chromatography and tandem mass spectrometry analysis revealed that OPN peptides derived from Alpl^−/− bone and osteoblasts yielded a higher proportion of phosphorylated peptides than samples from WT mice, and at least two phosphopeptides, p(S¹⁷⁴FQVS¹⁷⁸DEQY¹⁸²PDAT¹⁸⁶DEDLT¹⁹¹)SHMK and FRIp(S²⁹⁹HELES³⁰⁴S³⁰⁵S³⁰⁶S³⁰⁷)EVN, with one non-localized site each, appear to be preferred sites of TNAP action on OPN. Our data suggest that the pro-mineralization role of TNAP may be related not only to its accepted pyrophosphatase activity but also to its ability to modify the phosphorylation status of OPN.

Introduction

Tissue-nonspecific alkaline phosphatase (TNAP) is encoded by the ALPL gene in humans and the Alpl (a.k.a Akp2) gene in mice. Mouse TNAP is first expressed in the inner cell mass of blastocysts, primordial germ cells, the neural tube and placenta during embryogenesis⁽¹⁾, then in osteoblasts, chondrocytes, odontoblasts, renal tubule cells, macrophages, adipocytes and endothelial cells during later stages.⁽²⁾ We have previously generated Alpl knockout mice and demonstrated that they exhibit impaired bone mineralization and pyridoxine-dependent seizures that lead to death before weaning.^(2,3) The phenotype of the Alpl^−/− mouse, includes barely detectable plasma alkaline phosphatase (AP) activity, elevated plasma pyridoxal-5-phosphate (PLP; a form of vitamin B6) and inorganic pyrophosphate (PP_i), rickets/osteomalacia, and postnatal death, accurately modeling the infantile form of hypophosphatasia (HPP).^(4,5) TNAP is a GPI-anchored ectoenzyme capable of dephosphorylating a broad range of molecules in vitro such as p-nitrophenylphosphate, β-glycerophosphate (βGP), DNA, phosphoproteins, PP_i and PLP⁽⁶⁾ and there is conclusive evidence to indicate that the latter two molecules represent physiological substrates of TNAP, since the elevated plasma levels of PP_i and PLP in both Alpl^−/− mice and HPP patients explain the pathophysiology of HPP.⁽⁷⁾ Breeding Alpl^−/− mice to mice deficient in the production (Enpp1^−/−) or transport (ank/ank) of PP_i partially corrects the skeletal defect in Alpl^−/− mice confirming that increased PP_i levels are responsible for the skeletal disease seen in HPP.^(8,9) Similarly, pyridoxal supplementation of Alpl^−/− mice leads to prevention of the epileptic seizures, confirming the role of TNAP in the metabolism of PLP in vivo.^(3,10) Our recent data have shown that enzyme replacement therapy (ERT) using mineral-targeted recombinant TNAP prevents the skeletal and dental abnormalities associated with HPP in the Alpl^−/− model.^(11-14) The therapeutic principle involves administration of recombinant TNAP fused to a C-terminal polyaspartic acid sequence that confers high affinity for hydroxyapatite. This mineral-targeted TNAP acts at sites of mineralization to prevent the skeletal and dental defects by reducing local PP_i concentrations, and increasing levels of absorbable pyridoxal by dephosphorylating PLP to prevent seizures in HPP mice.⁽¹¹⁾

Alpl knockout mice also display marked changes in osteopontin (OPN, encoded by Spp1), with elevated expression at both its RNA and protein levels.^(15-17) OPN is expressed in a wide variety of cells, such as osteoblasts, chondrocytes, osteocytes, osteoclasts, nephrons, trophoblasts, T-lymphocytes, vascular smooth muscle cells, macrophages and certain cancer cells.^(18-20) While the biological role of OPN is incompletely understood, one known function is to anchor osteoclasts to the hydroxyapatite surface through its poly-aspartic acid sequences.⁽²¹⁾ OPN also binds to CD44 and α_vβ₃ integrin via its RGD sequence and mediates cell signaling and/or migration.⁽²²⁾ OPN is a phosphorylated glycoprotein, with 36 serine/threonine phosphorylation sites in the human protein.⁽²³⁾ This phosphorylation is functionally important as the inhibitory effect of OPN on mineral deposition was diminished if 84% of covalently bound phosphates were removed from OPN.⁽²⁴⁾ Phosphorylated OPN inhibits mineralization in vascular smooth muscle cells, while dephosphorylated OPN does not.⁽²⁵⁾ Certain phosphorylated OPN peptides are also capable of inhibiting hydroxyapatite formation in vitro⁽²⁶⁾ and cause dose-dependent inhibition of mineralization in cultured cells.⁽²⁷⁾ It is important to determine whether the OPN in Alpl^−/− mice is phosphorylated since increased phosphorylated OPN could contribute to the impaired bone mineralization.

Expression of TNAP precedes that of OPN during osteoblast maturation⁽²⁸⁾ and an interesting cross-talk between TNAP and OPN expression has been recognized in bone cells. Inorganic phosphate, a product of TNAP activity, induces OPN expression in cultured osteoblastic cells.⁽²⁹⁾ Mutant mice lacking OPN (Spp1^−/−) do not show an obvious bone phenotype⁽³⁰⁾, but they are resistant to ovariectomy-induced osteoporosis.⁽³¹⁾ We have previously shown that high plasma OPN levels accompany the increased extracellular PP_i levels in Alpl^−/− mice and that [Alpl^−/−; Spp1^−/−] double knockout mice, have a partial improvement of the hypomineralization found in Alpl^−/− mice. This indicates that increased OPN contributes to the impaired bone mineralization of Alpl^−/− mice⁽¹⁷⁾, although a mechanistic explanation for this effect is still lacking. Here, we have used genetic means to demonstrate that TNAP affects the phosphorylation status of OPN in vivo. In addition we show that in vivo overexpression of TNAP leads to increased bone mass.

Materials and Methods

Transgenic mice

We established a transgenic (Tg) mouse line, Col1a1-Tnap, expressing the human TNAP cDNA under control of the mouse Col1a1 promoter. The designation Tg (+/−) or (+/+) is used to denote hemizygosity and homozygosity for the transgene, respectively. Col1a1-Tnap^+/+ mice were bred to Alpl^−/− mice (MGI strain ID: Alpl^tm1Jlm)⁽²⁾, to generate [Col1a1-Tnap^+/−; Alpl^−/−] mice that express human TNAP under control of the osteoblast-specific Col1a1 promoter in an Alpl null background. Another Tg mouse line expressing human TNAP cDNA under control of a liver specific Apolipoprotein E promoter (ApoE-Tnap) was reported previously.⁽³²⁾ApoE-Tnap mice were also crossed to Alpl^−/− mice to produce [ApoE-Tnap^+/−; Alpl^−/−] mice. All animal studies were conducted with approval of the Animal Usage Committee of the Sanford Burnham Medical Research Institute, La Jolla, CA.

Collection of tissue samples, plasma analysis and histological studies

Mice were anesthetized by intraperitoneal injection of Avertin and blood was collected by cardiac puncture. Plasma levels of AP activity were measured using a previously reported method.⁽¹¹⁾ Tissue samples for histological analysis were fixed in 4% paraformaldehyde/PBS solution and processed as previously described.⁽²⁾ Bone tissues from adult mice were decalcified with 0.125 M EDTA/10% formalin in H₂O (pH 7.2) for five days after fixation, and processed for paraffin sectioning. Immunostaining was performed using a standard avidin-biotin complex protocol. Undecalcified bone sections were prepared using Acrylosin SOFT Infiltration and Embedding (DHM, Villa Park, IL, USA). PP_i levels were measured as we previously reported,⁽⁸⁾ and mouse OPN in plasma was measured with ELISA (Enzo, Plymouth Meeting, PA, USA), following the manufacturer's protocol.

Bone histomorphometric analysis

Bone samples were fixed in 4% paraformaldehyde/PBS and washed in 10%, 15% and 20 % sucrose PBS prior to cryo-embedding in hexane dry-ice bath. Undecalcified sections were prepared by following Kawamoto's film method.⁽³³⁾ To compare vertebrae bones from 4-month-old WT, Col1a1-Tnap^+/− and Col1a1-Tnap^+/+ male mice were analyzed. To examine compensation by the transgene expression in TNAP null mice, tibia samples from WT, Col1a1-Tnap^+/−, Alpl^−/−, and Col1a1-Tnap^+/−; Alpl^−/− mice were analyzed. Postnatal mice at day 16 were used since this stage is the survival limit for most of Alpl^−/− mice. Von Kossa/Van Gieson stained sections were scanned by ScanScopeXT system (Aperio, Vista, CA, USA), and images were analyzed by using the Bioquant Osteo software (Bioquant Osteoanalysis Co., Nashville, TN).

Alizarin red (Sigma, Saint Louis, MO, USA) and calcein (TCI, Toshima, Tokyo, Japan) were administered to postnatal mice with an interval of 48 hours by subcutaneous injection. Alizarin red was injected at day 7 (20 mg/kg body weight) and calcein was injected at day 9 (20 mg/kg body weight) to WT, Col1a1-Tnap^+/−, Alpl^−/−, and Col1a1-Tnap^+/−; Alpl^−/− mice, and the injected mice were collected at day 11. Coronal sections of undecalcified calvarial bones were prepared by Kawamoto's film method as same as the bone/osteoid analysis. Pictures of fluorescent images were taken with Nikon TE300 microscope, and distance between Alizarin red and calcein were measured by SPOT software (Diagnostic Instruments Inc., Sterling Heights, MI, USA). At least 10 independent positions were measured to obtain an average value per each animal.

Western blots

After careful removal of skin, tendons and muscle, leg bones were snap frozen with dry ice. All bone tissues were from four-month-old mice except Alpl^−/− samples, as these mice die at around postnatal day 20. Frozen bones were crushed into powder and suspended in lysis solution containing 4 M guanidine HCl, 50 mM Tris HCl, 0.5 M EDTA, 2 mM PMSF, 5 mg/L pepstatin and 1 mg/L soybean trypsin inhibitor (pH 7.5).⁽³⁴⁾ This guanidine-EDTA solution was also used to extract protein from mineralized osteoblast cultures. After rotation at 4°C for 48 hours, the solubilized bone samples were dialyzed against TBS solution containing 5 mM EDTA and 2 mM PMSF (pH 7.5) at 4°C to remove guanidine salt. Protein concentration was determined using a bicinchoninic acid assay kit according to manufacturer's instructions (Thermo Fisher Scientific Inc., Rockford, IL, USA). SDS PAGE was performed using a standard protocol, except that 1.7% SDS was used in the running buffer for bone samples to minimize the background, which is likely caused by OPN because of its absence in the samples from Spp1^−/− mice. Rat anti-human TNAP monoclonal antibody (R&D Systems, Minneapolis, MN, USA) and goat anti-mouse OPN antibody (Abcam, Cambridge, MA, USA) were used for detection of TNAP and OPN, respectively. Detection was followed by standard procedure using ECL Plus (GE Healthcare, Pittsburgh, PA, USA).

Phosphorylation analysis

Protein samples extracted with the guanidine/EDTA as described earlier were incubated with mouse anti-OPN antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight, and equally divided into two tubes. Protein A Sepharose (Amersham Pharmacia, Uppsala, Sweden) was added to each tube and incubated at 4°C overnight. Washed beads were resupended in 50 μL of the same buffer. Two microliters of 10 U/μL calf intestinal alkaline phosphatase (IAP) (New England Biolabs, Ipswich, MA, USA) were added into the first tube, while 2 μL of 10 mM Tris HCl (pH 8.2) was added to the second tube. After a 2 hr incubation under rotation at 37°C, reactions were stopped with sample buffer and heated at 95°C for 5 min prior to electrophoresis. Blotted membranes were stained with goat anti-OPN antibody (Abcam, Cambridge, MA, USA).

Samples extracted with guanidine/EDTA from osteoblast culture and bones were dialyzed against TBS containing 2mM PMSF to remove EDTA and loaded onto 10% acrylamide gels containing 30 M Phos-Tag [i.e. 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II)], a compound that binds to phosphorylated ions of phosphoproteins and lowers the migration speed (Wako Pure Chemical Industries, Ltd, Osaka, Japan).⁽³⁵⁾ Migration pattern of OPN was compared by western blots with goat anti-OPN antibody (Abcam, Cambridge, MA, USA).

For proteomics analysis, guanidine/EDTA extracts of leg bones and mineralized osteoblast culture were incubated with a combination of mouse and goat anti-OPN antibodies, and precipitated with Protein G agarose beads (Thermo Scientific, Rockford, IL USA). Samples eluted from the Protein G beads were subjected to TiO₂-based enrichment procedure. OPN phoshopeptides were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS) on Michrom MS2 HPLC-captive spray-LTQ Orbitrap Velos with ETD (Thermo Scientific, Rockford, IL USA). Analytical preparations and data processing were conducted by the Proteomics core facility at the Sanford-Burnham Medical Research Institute.

Micro-computed tomography and X-ray analysis

Skull bones, vertebrae and femur from five Col1a1-Tnap^+/+ and five wild-type (WT) control mice (four-month-old, two males and three females) were dissected and fixed in 4% paraformaldehyde/PBS, then analyzed using micro-computed tomography (μCT) by Numira Biosciences (Salt Lake City, UT, USA) using a high resolution, volumetric μCT scanner (μCT40, ScanCo Medical, Zurich, Switzerland). The image data was acquired with the following parameters: 10 μm (skull bones and vertebrae) and 6 μm (femur) isotropic voxel resolution at 300 ms exposure time, 2000 views and 5 frames per view. The μCT generated DICOM files were used to analyze the samples and to create volume renderings. The raw data files were viewed using Microview (GE Healthcare, Milwaukee, WI, USA) and AltaViewer software (Numira Biosciences). SCIRun (Scientific Computing and Imaging Institute, University of Utah) was used for cutaway images. X-ray images were obtained with a Faxitron MX-20 (Faxitron X-ray Corporation, Chicago, IL, USA) using energy of 20 kV.

Results

Correction of the HPP phenotype by tissue-directed expression of TNAP

Two transgenic models of TNAP overexpression were compared for their ability to prevent the HPP phenotype in Alpl^−/− mice. We generated a Col1a1-Tnap mouse strain, expressing TNAP under control of the Col1a1 promoter (Figure 1A, B). The Col1a1-Tnap mice appear healthy and both genders are fertile. Breeding of this mouse line to Alpl^−/− HPP mice demonstrated that expression of the Col1a1-Tnap transgene rescued the lethal phenotype of Alpl^−/− mice, and [Col1a1-Tnap^+/−; Alpl^−/−] animals live at least 20 months without evidence of epilepsy; however, their average body weight is reduced compared to WT littermate controls (Figure 1C). Plasma levels of TNAP were 527 ± 282 μg/mL in Col1a1-Tnap^+/− or Col1a1-Tnap^+/+, 35 ± 0.01 μg/mL in WT and 478 ± 148 μg/mL in [Col1a1-Tnap^+/−; Alpl^−/−] mice (Figure 1D). Expression of the Col1a1-Tnap transgene is restricted to osteoblasts of long bones and calvaria but not chondrocytes (Figure 2I, J). Other organs that express the Col1a1-Tnap transgene include the adrenal glands, spinal cord and fore brain (Figure 2J, L). Expression of Col1a1-Tnap transgene in bone was also confirmed at the RNA level by q-RTPCR (Figure S1).

Figure 1. Construction and characterization of Col1a1-Tnap mice.

(A) The 5.3 kb transgene fragment consists of a 2.3 kb Col1a1 promoter, an intron, a full TNAP cDNA (2.5 kb) and a polyA sequence isolated from the pStec1 backbone vector by Sac I digestion.⁽³⁶⁾ The 2.3 kb Col1a1 promoter was isolated from the pJ251 vector (a kind gift from Dr. Gerard Karsenty, Columbia University, NY) and the 2.5 kb fragment of human ALPL cDNA was derived from pSV2Aalp (ATCC 59634). The purified DNA construct was used for microinjection into the pronucleus of embryos from FVB/N mice in the transgenic facility at the Sanford-Burnham Medical Research Institute. (B) Integration of transgene was analyzed by Southern hybridization using standard protocols. Four micrograms of tail DNA was digested with Hind III and probed with 675 bp fragment from intron 2 of Alpl gene together with a 498 bp Bgl I fragment from the human ALPL cDNA. The transgene was transmitted to the offspring in a Mendelian pattern. (C) Body weight at four months of age: female Col1a1-Tnap^+/− or Col1a1-Tnap^+/+ (ColTg) vs WT p=0.5401, female WT vs [Col1a1-Tnap^+/−; Alpl^−/−] (ColTg; Alpl−/−) p=0.0276. The body weight difference is more pronounced in female than male animals. (D) An average TNAP value from triplicated measurement for each animal was plotted in the Fig. D, (numbers of animals, Col1a1-Tnap^+/−, Col1a1-Tnap^+/+, WT and Col1a1-Tnap^−/−, are n=18, 7, 11, 5 respectively). Plasma AP levels display a wide range but are consistently elevated in the transgenic mice. Col1a1-Tnap^+/− or Col1a1-Tnap^+/+: 504 ± 261 μg/mL, WT: 25.8 ± 14.7 μg/mL, [Col1a1-Tnap^+/− or Col1a1-Tnap^+/+; Alpl^−/−]: 478 ± 221 μg/mL. The difference between Col1a1-Tnap^+/− (hemizygous) and Col1a1-Tnap^+/+ (homozygous) was not significant (p=0.3967), while hemizygous Tg display a significant increase in AP compared to WT (p<0.0001).

Figure 2. AP activity staining on tissues from 11-day-old mice.

A, B, C and D: wild type. E, F, G and H: [ApoE-Tnap^+/−; Alpl^−/−]. I, J, K and L: [Col1a1-Tnap^+/−; Alpl^−/−]. M, N, O and P: Alpl^−/− mice. A, E, I and M: fibula. B, E, H and K: calvaria and forebrain. C, G, K and O: liver. D, H, L and P: kidney and adrenal gland. Organs for each genotype shown are derived from a single animal. Two to four mice per each genotype were analyzed and typical results were shown. (×100)

The second transgenic line expressing TNAP under control of the ApoE promoter, ApoE-Tnap, overexpresses TNAP in bone (Figure 2E) and in the liver and kidney (Figure 2G, H). It is noteworthy that TNAP is a well-known serum marker for liver dysfunction in humans,⁽³⁷⁾ while normal mouse liver expresses virtually no TNAP activity, except in the bile canaliculi in certain strains.^{(38, 39)} The TNAP plasma levels in ApoE-Tnap^+/− and ApoE-Tnap^+/+ mice were 11,026 ± 2,094 μg/mL and 23,102 ± 15,790 μg/mL, respectively. Expression of ApoE-Tnap transgene in the liver and bone was also confirmed by q-RTPCR (Figure S2).

Col1a1-Tnap^+/− and ApoE-Tnap^+/− mice did not show marked reduction of PP_i levels in the plasma compared to WT mice (Figure 3A, C). Plasma PP_i of [Col1a1-Tnap^+/−; Alpl^−/−] was slightly elevated, while levels from [ApoE-Tnap^+/−; Alpl^−/−] were normal, most likely due to the high TNAP expression in the liver and bones in ApoE-Tnap^+/− mice (Figure 2E, G).

Figure 3. PP_i and OPN measurement.

A and C: plasma PP_i. B and D: plasma OPN. A and B: four-month-old Col1a1-Tnap^+/−, WT and [Col1a1-Tnap^+/−; Alpl^−/−] mice. C: four-month-old ApoE-Tnap^+/−, WT and [ApoE-Tnap^+/−; Alpl^−/−] mice. D: 11-day-old Col1a1-Tnap^+/−, WT, [Col1a1-Tnap^+/−; Alpl^−/−] and Alpl^−/− mice. E: correlation between body weight and plasma OPN in 11-day-old Alpl^−/− mice. An average value from duplicated assay for each mouse was plotted in the figures. (A, B and C: n=5, D: n=4 except that Alpl^−/− was n=18).

Overexpression of TNAP leads to increased bone mineralization

We evaluated the ability of Col1a1-Tnap calvarial osteoblasts to mineralize in vitro. We tested both low and high concentrations of βGP (2 mM and 10 mM) as the phosphate source since high ®GP (10 mM) can cause non-physiological mineral deposits.⁽⁴⁰⁾ At both concentrations, increased mineralization was observed in the osteoblasts overexpressing the Col1a1-Tnap transgene (Figure 4) compared to control cells. [Col1a1-Tnap^+/−; Alpl^−/−] cells showed as much mineralization as heterozygous Alpl^+/− osteoblasts, while Alpl^−/− cells failed to mineralize in agreement with our previous data.⁽¹⁵⁾

Figure 4. Mineralization assay.

Osteoblasts were isolated from calvarial bones of two-day-old mice as described.⁽⁸⁾ Culture media containing 100 μg/mL ascorbic acid and 2 or 10 mM ®GP was renewed every second day and the cells were fixed in 100% ethanol 19 days later for AP staining and mineralization assay.⁽⁴¹⁾ A western blot to detect TNAP is shown in the top row. The sample from [Col1a1-Tnap^+/−; Alpl^−/−] (ColTg; Alpl-/-) represents human TNAP protein expressed from the transgene and the sample from Alpl^+/− control shows endogenous mouse TNAP. Human and mouse TNAP share 91.5 % homology in their peptide sequences, and the rat monoclonal antibody used in this study recognizes both human and mouse TNAP. Cells of each genotype were derived from a single newborn pup. The same experiment was repeated twice to test reproducibility.

Histomorphometric analysis of adult mice showed that vertebral bones from Col1a1-Tnap^+/− and Col1a1-Tnap^+/+ mice had higher values of BV/TV (bone area per tissue area) compared to WT controls as well as significantly reduced osteoid (Figure 5A). We conducted TRAP staining on adult tibiae and vertebrae bones. TRAP positive area per tissue volume (%) were 3.400 ± 1.049, 3.246 ± 0.2671, and 3.279 ± 0.3498 (%) for WT, ColTg(+) and ColTg(++) tibiae respectively, and 1.535 ± 0.6108, 1.566 ± 0.9479, and 1.456 ± 0.7771 (%) for WT, ColTg(+) and ColTg(++) L3 vertebrae, respectively. Alpl^−/− mice do not exhibit changes in osteoclasts numbers as we previously published ⁽¹⁵⁾, and we did not observe decreased osteoclast activity in the Cola1-Tnap transgenic mice. In tibia samples from 16-day-old mice, increase of mineralization was not significant in Col1a1-Tnap^+/− mice; however, poor mineralization in Alpl^−/− was improved by expression of human TNAP gene as increased BV/TV and reduced osteoid in Col1a1-Tnap^+/−, Alpl^−/− were observed (Figure 5B). The low mineral apposition rate in Alpl^−/− was improved by the transgene expression as shown in Figure 5C.

Figure 5. Bone histomorphometric analysis.

A: comparison of area of mineralized bone (BV/TV) and unmineralized osteoind (OV/BV) in L2 and L3 vertebrae bones from four-month-old WT, Col1a1-Tnap^+/−, and Col1a1-Tnap^+/+ mice (n=3). B: comparison of area of mineralized bone (BV/TV) and unmineralized osteoind (OV/BV) in tibia from 16-day-old WT, Col1a1-Tnap^+/−, Alpl^−/− and [Col1a1-Tnap^+/−; Alpl^−/−] mice (n=3). C: mineral apposition rates in parietal bones from 11-day-old WT, Col1a1-Tnap^+/−, Alpl^−/− and [Col1a1-Tnap^+/−; Alpl^−/−] mice (n=3)

We also compared the degree of bone mineralization of Col1a1-Tnap and ApoE-Tnap mice by μCT analysis. Measurements of bone surface per volume and trabecular thickness indicated increased bone formation in the Col1a1-Tnap^+/+ femur (Table 1, Figure S3). Furthermore, significant changes in the bone volume fraction, bone surface per volume, trabecular number, trabecular thickness and trabecular separation were observed in the L2 vertebrae (Table 1). The average structure model index of L2 bone in Col1a1-Tnap^+/+ and WT controls was 1.1824 and 1.9962, respectively. Similar results were obtained from analysis of femora and L2 vertebrae of ApoE-Tnap mice (four-month-old, two males and three females) (Table S1). In comparison to Col1a1-Tnap, ApoE-Tnap mice showed a more significant increase in femur than vertebrae, most likely due to the higher expression of TNAP in the trabecular osteoblasts.

Table 1.

MicroCT analysis of bones from Col1a1-Tnap Tg mice. BMD: Bone mineral density; BV/TV: Bone volume/Total volume; BS/BV: Bone surface/Bone volume; Tb.N: Trabecular number; Tb.Th: Trabecular thickness; Tb.Sp: Trabecular separation.

		WT	Col1a1-Tnap+/+	*
Distal femur	BMD	905.3 ± 22.64	931.3 ± 40.99	p = 0.3737
	BV/TV	0.05022 ± 0.01959	0.06546 ± 0.01097	p = 0.2552
	BS/BV	69.86 ± 4.886	58.36 ± 10.01	p = 0.0277
	Tb.N	1.731 ± 0.6117	1.885 ± 0.2661	p = 0.6493
	Tb.Th	0.02876 ± 0.002113	0.03504 ± 0.005694	p = 0.0401
	Tb.Sp	0.6111 ± 0.2235	0.5043 ± 0.07631	p = 0.4079
L2 vertebrae	BMD	873.5 ± 26.1	908.7 ± 17.15	p = 0.1241
	BV/TV	0.08462 ± 0.02315	0.1756 ± 0.03187	p = 0.0048
	BS/BV	63.94 ± 7.525	43.57 ± 43.57	p = 0.0020
	Tb.N	2.649 ± 0.4983	3.789 ± 0.4503	p = 0.0157
	Tb.Th	0.03164 ± 0.003711	0.04626 ± 0.004501	p = 0.0013
	Tb.Sp	0.3574 ± 0.07992	0.2206 ± 0.03194	p = 0.0176

^*p values were obtained by two-way ANOVA between WT and Col1a1-Tnap^+/+

TNAP affects the phosphorylation status of OPN in vivo

Expression of the TNAP transgene is significantly lower than endogenous TNAP (Figure S4A and B), while OPN was highly expressed in both [Col1a1-Tnap^+/−; Alpl^−/−] and WT. The unchanged plasma PP_i in adult [Col1a1-Tnap^+/−; Alpl^−/−] animals (Figure 3A) may indicate that the level of TNAP expressed in these osteoblasts was not high enough to affect the levels of systemic PP_i, and does not exclude the possibility of localized reduction of PP_i in the mineralizing microenvironment. The corresponding results in PP_i and OPN, unchanged in Col1a1-Tnap^+/− and increase in [Col1a1-Tnap^+/−; Alpl^−/−] shown in Figure 3A and 3B, concur with the previously reported strict correlation between plasma PP_i and OPN concentrations.⁽¹⁷⁾ OPN is a heavily phosphorylated extracellular matrix protein, and phosphorylated OPN inhibits mineralization. ^(24-27) We previously reported that OPN is upregulated in Alpl^−/− animals, although the phosphorylation status of the increased OPN in these mice was not determined.^(14,17) According to the algorithm NetPro 2, mouse OPN is predicted to have 43 phosphorylation sites. As the molecular weight of a phosphate group is 94.97, the total molecular weight of the 43 phosphate residues would be approximately 4 kDa. Thus, we expected that fully phosphorylated OPN would display a 4 kDa difference in SDS-PAGE compared to the fully dephosphorylated OPN, although we also anticipated that OPN signal will appear as a broad band due to the high degree of post translational modifications. First, we tested if OPN from Alpl^−/− osteoblasts exhibits larger molecular size than from control cells (Figure 6A). OPN from Alpl^−/− cells was detected in the larger molecular mass range (highly phosphorylated) while OPN from Alpl^+/− display a smaller molecular weight range, although all the bands appear quite broad. OPN from [Col1a1-Tnap^+/−; Alpl^−/−] cells appeared in the similar range as Alpl^+/−, and [Col1a1-Tnap^+/−; Alpl^+/−] cells showed an increased amount of OPN in the smaller range than Alpl^+/−. This result suggests that endogenous mouse TNAP dephosphorylates OPN in osteoblasts and that overexpressed human TNAP dephosphorylates OPN, compensating for the lack of endogenous TNAP in [Col1a1-Tnap^+/−; Alpl^−/−] cells. In bone extracts, the sizes of OPN were less changed possibly because of coexisting OPN originated from chondrocytes without transgenic expression of TNAP. However, dephosphorylated OPN is visible as a separate band smaller than the 52 kDa marker. [Col1a1-Tnap^+/−; Alpl^−/−] bones showed this band as well as WT and Col1a1-Tnap samples, while it was not observed in the Alpl^−/− bones, indicating that OPN is less dephosphorylated in the Alpl^−/− samples (Figure 6B).

Figure 6. Western blotting.

Thirty nanograms of recombinant mouse OPN (Sigma, Saint Louis, MO, USA) was digested with 10 U of IAP in AP reaction buffer for two hours and used as a control of dephosphorylated OPN (r.mOPN+IAP). A: cultured osteoblasts under mineralizing condition. B: bone tissue. The same membranes were re-probed for β-actin. C: Immunoprecipitation experiment. Untreated and IAP treated samples were compared by western blot with goat anti-OPN antibody (Figure 6C left and right). D: Comparison of OPN migration in Phos-Tag SDS PAGE. Left: osteoblast extracts. Right: bone extracts from 11-day-old and four-month-old. Western blots with anti-OPN antibody were shown along with Ponceau S staining of the same membrane to indicate the total protein blotted. Samples in each lane were derived from a single mouse and these experiments were repeated at least 2 times with samples derived from different animals.

OPN protein precipitated with a mouse monoclonal antibody was detected as a broad band ranging from 52 to 65 kDa, however, OPN precipitated from the Alpl^−/− sample is devoid of smaller size forms (Figure 6C left). After dephosphorylation by commercial bovine intestinal alkaline phosphatase (IAP), all signals shifted to the smaller range closer to 52 kDa marker (Figure 6C right). IAP-treatment of Alpl^−/− derived OPN reduced the size of the protein to be similar to the other samples. The pattern of OPN signal from [Col1a1-Tnap^+/−; Alpl^−/−] was not distinguishable from either WT or Col1a1-Tnap^+/− indicating that human TNAP is dephosphorylating OPN in vivo.

In SDS-PAGE containing Phos-Tag compound, migration speed decreases as the degree of phosphorylation of the protein increases. OPN from Alpl^−/− osteoblasts was not detected suggesting that this OPN form was highly phosphorylated and did not migrate after binding to Phos-Tag, while OPN from cells expressing Col1a1-Tnap mice contained OPN forms migrating faster than those in WT because of increased dephosphorylation (Figure 6D, left). Phos-Tag analysis of bones from 11-day-old mice showed only background signal in Alpl^−/− while a detectable band is seen in [Col1a1-Tnap^+/−; Alpl^−/−] bones. In adult bones, OPN in [Col1a1-Tnap^+/−; Alpl^−/−] showed signals relatively similar to the WT (Figure 6D, right).

OPN phosphopeptides were isolated from bones and cultured cells from each genotype, i.e., Col1a1-Tnap^+/−, WT, [Col1a1-Tnap^+/−; Alpl^−/−] and Alpl^−/− and analyzed by TiO₂-LC-MS/MS (Table 2). Most of phosphorlylation sites are non-localized except the one that comprises Ser287. The Alpl^−/− bones and osteoblasts contained increased numbers of OPN phosphopeptides. [Col1a1-Tnap^+/−; Alpl^−/−] osteoblasts showed changes in that pattern, while bone of [Col1a1-Tnap^+/−; Alpl^−/−] did not show reduction of phosphopeptides most likely due to the large amount of the chondrocytes expressing OPN but no TNAP. Two OPN peptides, p(S¹⁷⁴FQVS¹⁷⁸DEQY¹⁸²PDAT¹⁸⁶DEDLT¹⁹¹)SHMK and FRIp(S²⁹⁹HELES³⁰⁴S³⁰⁵S³⁰⁶S³⁰⁷)EVN, with one non-localized site each, likely contain preferred sites for TNAP-mediated dephosphorylation.

Table 2.

Phosphopeptide sequences obtained from TiO2-LC-MS/MS analysis. All phosphorylation sites are non-localized except the one contains Ser 287. Residue numbers were based on the OPN isoform NP_001191131. The bottom row contains the total number of the spectral counts of individual phosphopeptides in each genotype.

phosphopeptide sequence	Bone ColTg	Bone WT	Bone ColTg; Alpl−/−	Bone Alpl−/−	Ostb WT	Ostb ColTg; Alpl−/−	Ostb Alpl−/−
	Vp(T38DS40GS42S43EEKLYS49)LHPDPIATWL	21	0	7	1	0	0	0
	p(S174FQVS178DEQY182PDAT186DEDLT191)SHMK	0	53	91	84	0	0	18
p(S192HMKS196GES199KES202)LDVIPVAQLL	7	11	13	36	3	0	2
LEHp(S244KES247QES250)ADQSDVIDSQASSK	0	8	7	31	0	0	4
LVLDPK(pS287)KEDDR	29	39	59	31	7	9	38
FRIpp(S299HELES304S305S306S307)EVN	25	22	24	21	1	0	4
FRIp(S299HELES304S305S306S307)EVN	51	36	65	63	8	10	38
	133	169	266	267	19	19	100

Bone phenotype of [Col1a1-Tnap^+/−; Alpl^−/−]

Postnatal 11-day-old [Col1a1-Tnap^+/−; Alpl^−/−] mice, did not exhibit any apparent bone phenotype reminiscent of HPP. However, in the four-month-old adult animals, X-ray analysis revealed a widened tibial epiphysis and shorter tibial length (Figure 7A). Measurements of the greatest length between the medial malleolus and medial chondyle in WT and [Col1a1-Tnap^+/−; Alpl^−/−] were 18.75 ± 0.24 mm and 17.25 ± 0.69 mm, respectively (n=5, two females and three males, p=0.0037). Impaired mineralization was observed in the epiphysis of [Col1a1-Tnap^+/−; Alpl^−/−] mice (Figure 7B, E). Immunohistochemistry indicated that expression of the human TNAP in adult [Col1a1-Tnap^+/−; Alpl^−/−] mice was significantly low in the endochondral ossification sites (Figure S4). These observations indicate that human TNAP compensated for lack of endogenous mouse TNAP in the cortical bone, but the lack of TNAP in chondrocytes affected normal development during endochondral ossification despite the high circulating levels of TNAP.

Figure 7. Bone phenotype of [Col1a1-Tnap^+/−; Alpl^−/−].

A: X-ray image of hind limbs. [Col1a1-Tnap^+/−; Alpl^−/−] (left) and WT (right). B: Higher magnification of X-ray images of knee joints. Top [Col1a1-Tnap^+/−; Alpl^+/+], middle WT and bottom [Col1a1-Tnap^+/−; Alpl^−/−]. C, D and E are von Kossa staining on undecalcified femur sections from [Col1a1-Tnap^+/−; Alpl^+/+], WT and [Col1a1-Tnap^+/−; Alpl^−/−], respectively. F, G and H: WT control. I, J and K: [Col1a1-Tnap^+/−; Alpl^−/−]. F and I: distal femur (×40). G, H, J and K: proximal tibia (×400). F, G, I and J: Goldner's trichrome staining. H and K: immunohistochemistry showing expression of endogenous mouse TNAP in WT control (H) and human TNAP in [Col1a1-Tnap^+/−; Alpl^−/−] (K). G H, and J K are serial sections respectively. The epiphyseal plate is almost missing from some areas of the tibia and femur of [Col1a1-Tnap^+/−; Alpl^−/−] mice (Figure 7I). The proliferating chondrocyte zone has an abnormal structure (arrows in Figure 7J) and formation of trabecular bones is also reduced in the adult [Col1a1-Tnap^+/−; Alpl^−/−] mice (Figure 7J). Endogenous TNAP is highly expressed near the hypertrophic zone (Figure 7H), while the expression of transgenic TNAP is limited (Figure 7K).

Discussion

We reported the association between OPN expression and the pathophysiology of murine HPP in 2006,⁽¹⁷⁾ but a mechanistic interpretation of this association is still lacking. Our current data provide compelling evidence for the role of TNAP in determining the phosphorylation status of OPN, which in turn regulates its mineral binding properties. Our earlier work had shown that OPN expression is upregulated in the plasma and the skeleton of Alpl^−/− mice⁽¹⁵⁾, in agreement with the observation that OPN expression is increased in cultured fibroblasts from HPP patients.⁽⁴²⁾ Spp1^−/− mice show increased bone mineral density (BMD), despite the elevated extracellular PP_i that results from downregulated expression of Alpl and upregulated expression of Enpp1 and Ank.⁽¹⁷⁾ The HPP phenotype in Alpl^−/− mice was partially corrected in [Alpl^−/−; Spp1^−/−] double knockout mice, even though their elevated PP_i concentrations were unresolved, suggesting that increased OPN also contributes to the Alpl^−/− bone phenotype independently of the mineralization inhibitory action of PP_i.⁽¹⁷⁾ Several studies have shown that phosphorylated OPN exhibits an inhibitory effect on mineralization.^(24-26) Our current data show that OPN in the long bones of Alpl^−/− mice is highly phosphorylated and that the phosphorylation status of OPN is decreased by overexpression of human TNAP in the Alpl^−/− background. This is clear evidence that OPN represents a natural substrate for TNAP and extends the role of TNAP in skeletal mineralization to include not only P_i-generation from ATP^(43,45) and PP_i hydrolysis ^(8,9,32) but also modulation of OPN function, an important matricellular protein, by dephosphorylation.

Previous reports have shown that PP_i induces OPN expression in osteoblast cultures, indicating that increased local PP_i causes upregulation of OPN in Alpl^−/− bone.⁽⁹⁾ Our observations that both plasma PP_i and plasma OPN were unchanged in Col1a1-Tnap mice but elevated in [Col1a1-Tnap^+/−; Alpl^−/−] (Figure 3A, C) is in agreement with those earlier findings and support the strict correlation between plasma PP_i and OPN concentrations.⁽¹⁷⁾ In addition, Beck et al. reported that free phosphate produced by TNAP, using βGP as substrate, induced OPN expression in culture.⁽²⁹⁾ OPN expression in [Col1a1-Tnap^+/−; Alpl^+/−] was markedly higher than Alpl^+/− in an osteoblast culture containing excess βGP in agreement with that earlier report.⁽²⁹⁾ Thus, OPN expression is under control of the local P_i/PP_i ratio, which is known to influence skeletal mineralization in a skeletal site-specific manner.^(5,32)

An interesting sideline observation made during our work is that while most Alpl^−/− mice exhibit normal or only slightly smaller weight at age 11 days, we have occasionally observed some exceptionally small Alpl^−/− animals with poor response to vitamin B6 administration.⁽³⁾ As shown here, those Alpl^−/− mice with small body sizes tend to have highly elevated plasma OPN. These data indicate that Spp1 may act as a modifier gene for HPP, a disease that is notorious for the variable penetrance and severity of presentation.⁽⁴⁴⁾ Thus Spp1 joins ENPP1 and PHOSPHO1 as genes with the potential to modify the severity of presentation of HPP.⁽⁴⁵⁾

Another observation worth noting is that the [Col1a1-Tnap^+/−; Alpl^−/−] mice were smaller and their average body weight was lower than WT controls. This can be explained by their shortened long bones. Histological analysis revealed abnormal growth plate morphology in the [Col1a1-Tnap^+/−; Alpl^−/−] mice, indicating that despite TNAP being expressed in osteoblasts and that plasma TNAP levels were elevated in these mice, the growth plates were still affected by the lack of TNAP. This observation is important because it reinforces the rationale for using mineral-targeting TNAP for enzyme replacement therapy of HPP^(11-14) rather than soluble recombinant TNAP, which had proven ineffective in the past.^(46-49) Even though the transgenic expression of TNAP in osteoblasts rescues the lethal phenotype of Alpl deficiency, residual disease can be demonstrated in these animals. This is in sharp contrast to the robustness of the treatment with mineral-targeting TNAP, which reaches notoriously avascular sites such as the enamel organ.⁽¹⁴⁾ These data provide additional evidence to indicate that indeed TNAP must be able to act locally at sites of initiation of mineralization, and that increasing the levels of circulating TNAP are not sufficient for a complete therapeutic response.