Ionomycin ameliorates hypophosphatasia via rescuing alkaline phosphatase deficiency-mediated L-type Ca2+ channel internalization in mesenchymal stem cells.

The loss-of-function mutations in the ALPL result in hypophosphatasia (HPP), an inborn metabolic disorder that causes skeletal mineralization defects. In adults, the main clinical features are early loss of primary or secondary teeth, osteoporosis, bone pain, chondrocalcinosis, and fractures. However, guidelines for the treatment of adults with HPP are not available. Here, we show that ALPL deficiency caused a reduction in intracellular Ca2+ influx, resulting in an osteoporotic phenotype due to downregulated osteogenic differentiation and upregulated adipogenic differentiation in both human and mouse bone marrow mesenchymal stem cells (BMSCs). Increasing the intracellular level of calcium in BMSCs by ionomycin treatment rescued the osteoporotic phenotype in alpl+/− mice and BMSC-specific (Prrx1-alpl−/−) conditional alpl knockout mice. Mechanistically, ALPL was found to be required for the maintenance of intracellular Ca2+ influx, which it achieves by regulating L-type Ca2+ channel trafficking via binding to the α2δ subunits to regulate the internalization of the L-type Ca2+ channel. Decreased Ca2+ flux inactivates the Akt/GSK3β/β-catenin signaling pathway, which regulates lineage differentiation of BMSCs. This study identifies a previously unknown role of the ectoenzyme ALPL in the maintenance of calcium channel trafficking to regulate stem cell lineage differentiation and bone homeostasis. Accelerating Ca2+ flux through L-type Ca2+ channels by ionomycin treatment may be a promising therapeutic approach for adult patients with HPP.


INTRODUCTION
A loss-of-function mutation in the liver/bone/kidney alkaline phosphatase (ALPL) gene results in the life-threatening disease hypophosphatasia (HPP) during early developmental periods; HPP is characterized by hypomineralization of the skeleton and teeth. 1,2 Adult patients with HPP showed early loss of primary or secondary teeth, osteoporosis, bone pain, chondrocalcinosis, and fractures. Our previous study found age-related bone mass loss and marrow fat gain in heterozygous Alpl +/− mice. 3 Bone marrow mesenchymal stem cells (BMSCs) are multipotent cells capable of differentiating into various cell lineages, including osteoblasts and adipocytes. As age increases, BMSCs are more inclined to undergo differentiation into adipocytes rather than osteoblasts, resulting in an increased number of adipocytes and a decreased number of osteoblasts, which leads to osteoporosis. 4 Our previous study also showed that ALPL governed the osteo-adipogenic balance in BMSCs and prevented cell senescence. 3 ALPL is a ubiquitous plasma membrane-bound enzyme (ectoenzyme) that functions at physiological (neutral) and alkaline pH to hydrolyze several different molecules, including inorganic pyrophosphate, 5 pyridoxal-5-phosphate (the active form of vitamin B6), 6 and nucleotides. [7][8][9] However, the detailed mechanism of ALPL causing age-related osteoporosis is largely unknown.
In severely affected infants with HPP, hypercalcemia, and hypercalciuria are often reported as symptoms. 10,11 However, it is still not clear why calcium metabolism abnormalities are induced by an ALPL mutation, since ALPL plays an important role in generating inorganic phosphate. Meanwhile, whether aberrant calcium metabolism is involved in age-related osteoporosis in heterozygous Alpl +/− mice is also unclear. It is well accepted that calcium metabolism abnormalities are closely related to calcium channels on the cell surface. Calcium influx is controlled by voltage-gated Ca 2+ channels (VGCCs) or agonist-dependent and voltage-independent Ca 2+ entry pathways, which are called 'storeoperated' Ca 2+ channels (SOCs). Changes in intracellular Ca 2+ concentration ([Ca 2+ ] i ) play an essential role in regulating motility, apoptosis, differentiation, and many other cellular processes. 12 Aberrant intracellular [Ca 2+ ] i leads to the loss of Ca 2+ homeostasis, which causes abnormal calcium metabolism and bone disorders. 13,14 Several types of Ca 2+ channels are reported to regulate Ca V 3.3, as Ca V 1.4 expression seems to be restricted to the retina. The results showed that the total protein expression of Ca V 1.2 and Ca V 1.3 was decreased significantly in alpl +/− BMSCs compared with WT BMSCs (Fig. 1d). Total protein expression of Ca V 1.1, Ca V 2.2, Ca V 2.3, and Ca V 3.3 was not changed (Figs. 1d and S2e). However, total and membrane expression levels of Ca V 2.1, Ca V 3.1, and Ca V 3.2 were increased in alpl +/− BMSCs compared with WT BMSCs (Fig. S2e, f). The expression of Ca V 2.1, Ca V 3.1, and Ca V 3.2 in the cytoplasm was not changed significantly in alpl +/− BMSCs compared with that of WT BMSCs (Fig. S2g). We used a plasma membrane protein extraction kit (Abcam, ab65400) to isolate plasma membrane protein, and then we measured the expression levels of membrane Ca V 1.1, Ca V 1.2, and Ca V 1.3. The results showed that alpl +/− BMSCs expressed similar levels of Ca V 1.1 in the membrane and cytoplasm to what was observed in WT BMSCs (Fig. 1e). Considering the decreased Ca 2+ influx in alpl +/− BMSCs, we focused on Ca V 1.2 and Ca V 1.3. Membrane expression of calcium channels affects calcium influx, and we compared the expression of Ca V 1.2 and Ca V 1.3 in the membrane and in the cytoplasm. The results showed that the expression levels of membrane, Ca V 1.2 and Ca V 1.3 were decreased in alpl +/− BMSCs, but the cytoplasmic levels did not change (Fig. 1e). We also measured KCl-induced Ca 2+ influx after knockdown of Ca V 1.2 or Ca V 1.3 in WT BMSCs. The results showed that knockdown of Ca V 1.2 or Ca V 1.3 reduced KCl-induced Ca 2+ influx in WT BMSCs (Fig. S2h). Moreover, we labeled the surface molecules with biotin and used neutravidin ultralink resin beads to capture biotinylated surface proteins. Then, we used western blot analysis to show the expression levels of proteins captured by anti-Ca V 1.2 and Ca V 1.3 antibodies. The results showed that the amount of Ca V 1.2 or Ca V 1.3 on the cell surface was decreased in alpl +/− BMSCs compared with that of the WT BMSCs (Fig. 1f). However, overexpression of ALPL increased the expression of Ca V 1.2 and Ca V 1.3 on the membrane, as assayed by western blot (Fig. 1g). We also investigated the membrane localization of Ca V 1.2 and Ca V 1.3 in WT and alpl +/− BMSCs by confocal laser scanning microscopy. The results showed that Ca V 1.2 (FITC-labeled) and Ca V 1.3 (FITClabeled) were localized to the membrane (as visualized by CellMask™ Deep Red Plasma Membrane Stain) of WT BMSCs (Fig.  1h, i). However, Ca V 1.2 and Ca V 1.3 were absent from the membrane of alpl +/− BMSCs. Ca V 1.2 and Ca V 1.3 were localized to the cell membrane after overexpression of ALPL in alpl +/− BMSCs (Fig. 1h, i). These results suggest that ALPL modulates the expression of L-type Ca 2+ channels, especially Ca V 1.2 and Ca V 1.3.
ALPL-regulated MSC osteogenic/adipogenic lineage differentiation via the L-type Ca 2+ channel We knocked down ALPL in BMSCs by siRNA treatment (Fig. S3a) and confirmed that ALPL deficiency decreased the osteogenic differentiation and increased the adipogenic differentiation of BMSCs ( Fig. S3b-e). To explore the function of long-lasting voltage-gated calcium channel (L-VGCC) in the osteogenic/ adipogenic differentiation of BMSCs, we used L-type Ca 2+ channel blockers, diltiazem, or nifedipine, to treat WT BMSCs, and we explored their resulting osteogenic and adipogenic differentiation capacities ( Fig. S3f-i). We found that both nifedipine and diltiazem inhibited the osteogenic differentiation ability of BMSCs, as evidenced by decreases in mineralized nodule formation and in expression of the osteogenic markers Bglap, Ibsp, RUNX2, and Sp7. In addition, nifedipine promoted the adipogenic differentiation of BMSCs, whereas diltiazem inhibited the adipogenic differentiation of BMSCs, as assessed by oil red O staining. The western blot assay data showed the same changes in the adipogenic regulators PPARγ2 and LPL under adipogenic culture conditions. To further address whether the defect in osteogenic/adipogenic lineage differentiation in ALPL-deficient BMSCs was due to the abnormal membrane expression of VGCCs, especially Ca V 1. 2 Fig. 1 ALPL deficiency caused decreased membrane expression of L-type Ca 2+ channels in BMSCs. a Ca 2+ imaging showed decreased Ca 2+ influx in cultured alpl +/− BMSCs and WT BMSCs transfected with shALP (shALP/WT) after they were stimulated with 30 mmol·L −1 KCl for 3 min (n = 10). b No KCl-induced Ca 2+ influx was detected in cultured WT, alpl +/− , and shALP/WT BMSCs treated with 10 mmol·L −1 EGTA for 3 min (n = 10). c ALPL overexpression was mediated by a lentivirus in alpl +/− (Lenti-alp/alpl +/− ) BMSCs and resulted in an elevated Ca 2+ influx following stimulation with 30 mmol·L −1 KCl for 3 min (n = 10). d, e The expression of Ca V 1.  (Fig. 2f, g). However, knockdown of Ca V 1.2 or Ca V 1.3 (by treatment with siCa V 1.2 or siCa V 1.3, respectively) (Fig. S3l, m) in WT BMSCs caused decreased osteogenic and increased adipogenic differentiation ( Fig. 2h-k). These data indicate that ALPL regulates osteogenic and adipogenic lineage differentiation through Ca V 1.2and Ca V 1.3-mediated calcium influx.
ALPL deficiency promoted the internalization of L-type Ca 2+ channels in BMSCs To determine whether a lack of ALPL leads to channel internalization resulting in decreased membrane expression of Ltype Ca 2+ channels in BMSCs, we disrupted endocytosis by expressing a dominant-negative mutant of dynamin 1 (DN-Dyn1), which is a GTPase required for the formation of endocytic vesicles from the plasma membrane. 22 The expression of DN-Dyn1 in alpl +/− BMSCs prevented the loss of Ca V 1.2 and Ca V 1.3 on the cell surface ( Fig. 3a, b), providing evidence that a lack of ALPL causes internalization of the channels. Western blot analysis showed that    the expression levels of membrane Ca V 1.2 and Ca V 1.3 in alpl +/− BMSCs were increased after DN-Dyn1 transfection. However, the expression levels of cytoplasmic Ca V 1.2 and Ca V 1.3 in alpl +/− BMSCs were decreased after DN-Dyn1 transfection (Fig. 3c). The expression of DN-Dyn1 also prevented the decrease in KClinduced Ca 2+ influx in alpl +/− BMSCs (Fig. 3e). We next measured the time course of ALPL-dependent L-type Ca 2+ channel internalization. To study this process in live BMSCs, we used Dio to label the cell membrane (FITC-labeled) and constructed a plasmid to express Ca V 1.2 (DsRed-Cav1.2) to use in the transfection of cells. We recorded colocalization regions as region of interest (ROI) to record the time-course change of intensity of red fluorescence, and we quantified the red florescence in each ROI over a time-course lapse at 300 s minus 0 s and 600 s minus 0 s to determine changes during the process. The DsRed-Ca V 1.2 signal in alpl +/− BMSCs declined significantly after 10 min compared with that of the WT, DN-Dyn1-transfected alpl +/− BMSCs, and ALPL-overexpressed alpl +/− BMSCs, reflecting the decreased membrane expression of the L-type Ca 2+ channel (Fig. 3d, f and g). We also selected images at 0 s and 570 s to show the colocalization of DsRed-Ca V 1.2 with the cell membrane of BMSCs. Almost no region of colocalization was found in alpl +/− BMSCs ( Fig. 3h-k), which suggested that ALPL deficiency promoted the internalization of L-type Ca 2+ channels. To determine whether the expression of DN-Dyn1 rescues differentiation in ALPL-deficient BMSCs, we performed osteogenic and adipogenic induction after transfection. Expression of DN-Dyn1 increased osteogenic differentiation of alpl +/− BMSCs, as evidenced by increased mineralized nodule formation and expression of the osteogenic markers RUNX2 and Sp7 (Fig. S4a, b). In contrast, expression of DN-Dyn1 decreased adipogenic differentiation of alpl +/− BMSCs, as assessed by oil red O staining, which showed decreased numbers of adipocytes, and western blotting, which showed downregulation of the adipogenic regulators PPARγ2 and LPL under adipogenic culture conditions (Fig. S4c, d).
ALPL deficiency promoted the internalization of L-type Ca 2+ channels via binding to α2δ subunits Given that ALPL has been reported to hydrolyze inorganic pyrophosphate (PPi) and adenosine triphosphate (ATP), we compared the expression of Ca V 1.2 and Ca V 1.3 in BMSCs and BMSCs treated with PPi or ATP. However, the addition of exogenous PPi or ATP hardly changed the membrane expression of Ca V 1.2 and Ca V 1.3 in BMSCs (Fig. S5a). To further explore the molecular mechanism of ALPL-regulated internalization of Ca V 1.2 and Ca V 1.3, we measured the membrane localization of ALPL and calcium channels. We found that Ca V 1.2 and Ca V 1.3 overlapped with ALPL in BMSCs (Fig. 4a, upper panel), suggesting the association of ALPL and L-type Ca 2+ channels. However, no membrane location of Ca V 1.2 and Ca V 1.3 was found in alpl +/− BMSCs (Fig. 4a, lower panel). Several regions of ALPL and Ca V 1.2 and Ca V 1.3 colocalization were found in the cytoplasm of alpl +/− BMSCs (Fig. 4a, lower panel), indicating that ALPL may bind L-type Ca 2+ channels in the cytoplasm. We also used immunoprecipitation to confirm the association of ALPL and the L-type Ca 2+ channel. Immunoprecipitation using a control antibody did not isolate either protein, but immunoprecipitation with anti-ALPL resulted in coimmunoprecipitation with Ca V 1.2 or Ca V 1.3 (Fig. 4b).
In addition, we found that anti-Ca V 1.2 or anti-Ca V 1.3 immunoprecipitated ALPL (Fig. 4c), suggesting that ALPL associates with Ca V 1.2 and Ca V 1.3 in BMSCs.
We next investigated which regions of Ca V 1.2 and Ca V 1.3 are important for ALPL-regulated internalization. The α 2 δ subunit has been reported to traffic α1 subunits, which influences internalization of the channels. 23,24 Floxed alpl mice with Prrx1::Cre mice were crossed to generate early embryonic MSC-specific (Prrx1alpl −/− ) conditional alpl knockout mice (Fig. S5b, c). We isolated BMSCs from the Prrx1-alpl −/− mice (ALPL −/− ) and control alpl f/f littermates (control). Alpl −/− BMSCs showed decreased expression of ALPL, and treatment with a lentivirus that overexpressed ALPL (Lenti-alp) elevated the membrane expression of ALPL (Fig. S5d). To explore whether ALPL interacted with α 2 δ to regulate the internalization of Ca V 1.2 and Ca V 1.3, we expressed ALPL with α 2 δ or a mutant α 2 δ in alpl −/− BMSCs and examined the membrane expression of Ca V 1.2 and Ca V 1.3. alpl −/− BMSCs were isolated from Prrx1-alpl −/− mice and showed no expression of ALPL, Ca V 1.2, or Ca V 1.3 at the membrane (Fig. 4d). The membrane expression of Ca V 1.2 and Ca V 1.3 was increased, and ALPL was colocalized with Ca V 1.2 and Ca V 1.3 after transfection with ALPL and α 2 δ (Fig. 4d). However, the membrane expression of Ca V 1.2 and Ca V 1.3 was not increased, and ALPL was not colocalized with Ca V 1.2 and Ca V 1.3 after transfection with ALPL and mutant α 2 δ (Fig. 4d). Western blot analysis also confirmed that the membrane expression of Ca V 1.2 and Ca V 1.3 was increased after transfection with ALPL and α 2 δ (Fig. 4e). However, no membrane expression of Ca V 1.2 and Ca V 1.3 was found in alpl −/− BMSCs and alpl −/− BMSCs transfected with ALPL and mutant α 2 δ (Fig. 4e). Immunoprecipitation using a control antibody did not isolate either protein in WT or alpl −/− BMSCs, but immunoprecipitation with anti-α2δ in alpl f/f BMSCs isolated ALPL (Fig. 4f). To confirm that ALPL interacts with α 2 δ subunits and thus regulates the lineage differentiation of BMSCs, we transfected alpl +/− BMSCs with α 2 δ or mutant α 2 δ and assessed their osteogenic or adipogenic induction. alpl +/− BMSCs transfected with mutant α 2 δ showed decreased osteogenic differentiation and increased adipogenic differentiation compared with alpl +/− BMSCs transfected with α 2 δ ( Fig. S5e-h).
ALPL deficiency caused aberrant lineage differentiation of BMSCs through the Wnt/β-catenin pathway To examine how ALPL deficiency-induced reduction of Ca 2+ influx affects the osteogenic and adipogenic differentiation of BMSCs, we analyzed three Ca 2+ downstream pathways (PKC/Erk, PI3K/Akt/ GSK3β, and CaMKII/calcineurine A), which are closely linked to Ca 2 + -associated regulation of osteogenic differentiation. We found that the expression level of p-Akt significantly decreased along with the reduction of p-GSK3β in alpl +/− BMSCs and BMSCs transfected with shALP ( Fig. 5a). However, the PKC/Erk and CaMKII/calcineurine A pathways were not changed significantly in alpl +/− BMSCs or in BMSCs transfected with shALP compared with that of WT BMSCs (Fig. S6a). Because the decrease in GSK3β phosphorylation inhibits the nuclear translocation of β-catenin, which regulates the osteogenic and adipogenic differentiation of BMSCs, we examined the expression levels of total and active β-catenin. We found that the expression of active β-catenin was decreased in both alpl +/− BMSCs and BMSCs transfected with shALP (Fig. 5a). Moreover, when we overexpressed ALPL in alpl +/− BMSCs, the expression of p-Akt, p-GSK3β, and active β-catenin was increased to levels similar to those of the WT BMSCs (Fig. 5b). When we overexpressed Ca V 1.2 or Ca V 1.3 in alpl +/− BMSCs, the expression of p-Akt, p-GSK3β, and active β-catenin was increased (Fig. 5c). In addition, the expression of DN-Dyn1 in alpl +/− BMSCs also increased the expression of p-Akt, p-GSK3β, and active β-catenin compared with alpl +/− BMSCs (Fig. 5d). Taken together, these data indicate that ALPL regulates Ca 2+ influx to affect p-Akt and p-GSK3β expression and subsequently targets the Wnt/β-catenin pathway in BMSCs.
To further determine whether ALPL regulates osteogenic and adipogenic differentiation through the Akt/GSK3β/Wnt/β-catenin pathway, we used activators of Akt phosphorylation (sc79) and GSK3β phosphorylation (LiCl) (Fig. S6b) to treat alpl +/− BMSCs, and we found that sc79 and LiCl treatment increased osteogenic differentiation, as evidenced by increases in mineralized nodule formation and in expression of RUNX2 and Sp7 (Fig. 5e, f). In contrast, sc79 and LiCl treatment decreased adipogenic differentiation of alpl +/− BMSCs, as assessed by oil red O staining, which showed a decreased number of adipocytes, and western blotting, which indicated a downregulation of PPARγ2 and LPL under ALP regulates Ca2+ channel in BMSCs B Li et al.
Raising the intracellular level of calcium by ionomycin rescued ALPL deficiency-induced age-related osteoporosis Ionomycin was reported to cause a robust increase in Ca 2+ influx and an inhibition in calcium channel endocytosis. 25 Therefore, we treated 12-week-old alpl +/− mice with ionomycin intraperitoneally at a dose of 1 mg·kg −1 per day for 28 days. We confirmed that ionomycin treatment increased the calcium influx and the membrane expression of Ca V 1.2 and Ca V 1.3 in alpl +/− BMSCs (Fig. S7a, b). MicroCT and histological analyses showed that bone mineral density (BMD), Bone volume relative to tissue volume (BV/ TV), and distal femoral trabecular bone number (Tb.N) in 3-monthold alpl +/− mice were markedly decreased compared with that of the control WT littermates (Fig. 6a, b). We treated alpl +/− mice with ionomycin, which caused a robust rise in Ca 2+ influx in cells. 25 MicroCT and histological analyses showed that BMD, BV/ TV, and Tb.N in 3-month-old alpl +/− mice treated with ionomycin was markedly increased compared with that in alpl +/− mice (Fig.  6a, b). To observe changes in osteogenic/adipogenic lineage differentiation in vivo, we examined the number of adipocytes in the bone marrow of WT, alpl +/− mice, and alpl +/− mice treated with ionomycin. Interestingly, oil red O staining showed that the number of adipocytes in alpl +/− mouse bone marrow was markedly increased compared with that of WT littermates (Fig.  6c), indicating that alpl deficiency increased adipogenic lineage differentiation. However, the number of adipocytes in alpl +/− mouse bone marrow after ionomycin treatment was markedly decreased compared with that of alpl +/− mice (Fig. 6c). To confirm that alpl deficiency directly contributes to decreased osteogenesis, a calcein double labeling analysis was used to show a decreased bone formation rate in alpl +/− mice and an elevated bone formation rate in alpl +/− mice treated with ionomycin (Fig. 6d). Moreover, we found that the intracellular level of Ca 2+ in alpl +/− BMSCs was decreased compared with that of WT BMSCs, and ionomycin treatment elevated the intracellular level of Ca 2+ in alpl +/− BMSCs (Fig. 6e). We observed impaired osteogenic differentiation and increased adipogenic differentiation in alpl +/ − BMSCs, as evidenced by decreased mineralized nodule formation and an elevated numbers of adipocytes (Fig. 6f, h). The decreased expression of the osteogenic markers RUNX2 and Sp7 and increased expression of the adipogenic regulators PPARγ2 and LPL were shown by western blotting (Fig. 6g, i). Ionomycin treatment increased the osteogenic differentiation and decreased adipogenic differentiation of alpl +/− BMSCs (Fig. 6f- Cav1.3 and Tb.N in 3-month-old Prrx1-alpl −/− mice, and we found that they were markedly decreased compared with that of their control alpl f/f littermates (Fig. 7a, b). Floxed alpl littermates (alpl f/f ) were used as controls. MicroCT and histological analyses showed that, BMD, BV/TV, and Tb.N in 3-month-old Prrx1-alpl −/− mice treated with ionomycin were markedly increased compared with what was observed in the Prrx1-alpl −/− mice (Fig. 7a, b). To further detect changes in osteogenic/adipogenic lineage differentiation in BMSCs, we examined the number of adipocytes in the bone marrow of alpl f/f , Prrx1-alpl −/− mice, and Prrx1-alpl −/− mice treated with ionomycin. Oil red O staining showed that the number of adipocytes in Prrx1-alpl −/− bone marrow was markedly increased compared with that in the control alpl f/f littermates (Fig. 7c). However, the number of adipocytes in Prrx1-alpl −/− bone marrow after ionomycin treatment was markedly decreased compared with that of the Prrx1-alpl −/− mice (Fig. 7c). Calcein double labeling analysis showed a decreased bone formation rate in Prrx1-alpl −/− mice relative to that of control alpl f/f mice (Fig. 7d). Ionomycin treatment reversed the impaired osteogenesis in Prrx1alpl −/− mice. Moreover, the serum levels of RANKL and OPG were not significantly changed, as assessed by ELISA (Fig. S7c, d), suggesting that osteoclasts may not be altered in Prrx1-alpl −/− mice. The intracellular level of Ca 2+ in alpl −/− BMSCs was decreased compared with that in the control BMSCs, and ionomycin treatment elevated the intracellular level of Ca 2+ in alpl −/− BMSCs (Fig. 7e). In addition, BMSCs from Prrx1-alpl −/− mice showed decreased osteogenic differentiation and increased adipogenic differentiation compared with BMSCs from alpl f/f mice   ( Fig. 7f-i). BMSCs from Prrx1-alpl −/− mice treated with ionomycin showed increased osteogenic and decreased adipogenic differentiation ( Fig. 7f-i). These results indicate that ALPL deficiency in BMSCs induces an age-related osteoporosis phenotype and that ionomycin treatment reversed this phenotype.
ALPL deficiency promoted the internalization of L-type Ca 2+ channels in HPP patient-derived BMSCs We also collected bone marrow BMSCs from two HPP patients with mutations in the ALPL gene (A1 and A2, Table S1). The expression of ALPL on the membrane and cytoplasm was decreased in BMSCs from the two HPP patients compared with normal human bone marrow BMSCs (control) (Fig. 8a). We further determined whether the lack of ALPL resulted in decreased KClinduced Ca 2+ influx in A1 and A2 BMSCs. KCl-induced Ca 2+ influx was significantly decreased in culture-expanded A1 and A2 BMSCs compared with that of control BMSCs (Fig. 8b), and overexpression of ALPL elevated KCl-induced Ca 2+ influx (Fig. 8c, d) in A1 and A2 BMSCs. Moreover, we also found that the expression of DN-Dyn1 prevented the decrease in KCl-induced Ca 2+ influx in A1 and A2 BMSCs (Fig. 8c, d). The overexpression of ALPL or expression of DN-Dyn1 in A1 and A2 BMSCs increased the membrane expression of Ca V 1.2 and Ca V 1.3, as shown by confocal images (Fig. 8e), suggesting that ALPL deficiency causes channel internalization of Ca V 1.2 and Ca V 1.3 in a human model. To confirm that ALPL regulates the lineage differentiation of BMSCs, we overexpressed ALPL in A1 and A2 BMSCs and assessed their osteogenic or adipogenic ability after induction. A1 and A2 BMSCs transfected with the ALPL vector showed increased osteogenic differentiation and decreased adipogenic differentiation (Fig.  S8a-d). All of the above data show that ALPL regulates lineage differentiation of MSCs through association with the α2δ subunit of L-type Ca 2+ channels and through inhibiting the internalization of L-type Ca 2+ channels, thus increasing Ca 2+ influx (Fig. S9).

DISCUSSION
The deficiencies caused HPP are currently treated by bone anabolic and/or enzyme replacement strategies. Bone anabolic treatment, such as treatment with recombinant human parathyroid hormone analogs yielded debatable efficacy. 26 Asfotase alfa (Strensiq, Alexion), a bone-targeted enzyme replacement therapy, was approved for the long-term treatment of pediatric-onset HPP in the United States, Europe, Canada, and Japan. However, there are no guidelines for selecting adult patients for treatment, evaluating the results of treatment, or determining the optimal duration of treatment at this time. Patients with HPP can also develop secondary osteoporosis, bone marrow edema, and delayed fracture healing or difficulties with implant failure. 26 Thus, there is an urgent need to identify further bone-targeted treatment options for adult HPP patients.
Our previous study showed that the Alpl deficiency results in premature bone aging characterized by bone mass loss and simultaneous marrow fat gain. Although a pivotal role for ALPL in skeletal matrix mineralization has been established, the mechanism of ALPL regulating BMSC differentiation remains uncertain. Hypercalcemia was found in severely affected infants with HPP, which suggests that ALPL may modulate calcium homeostasis. In this study, we found that raising the intracellular level of calcium in BMSCs by ionomycin rescued ALPL deficiencyinduced age-related osteoporosis, which suggests that targeting calcium channels is a new approach for adult HPP treatment. Moreover, our study showed a new function for ALPL in controlling Ca 2+ influx by regulating the internalization of calcium channels, which balanced the osteogenic and adipogenic differentiation of BMSCs.
Voltage-dependent calcium channels are an important route by which Ca 2+ enters cells upon membrane depolarization to regulate calcium homeostasis. A previous study showed that VGCCs in BMSCs and osteoblasts regulate bone formation and that manipulating VGCCs promotes bone repair. 27,28 Of note, the L-VGCC, a major channel of calcium influx, is a part of the highvoltage activated family of VGCCs. L-type calcium channels are considered to play an important role in regulating BMSC   The involvement of ALPL in channel internalization leads to a change in the Ca V 1.2 and Ca V 1.3 number at the surface of cells. Several other proteins have been found to regulate the membrane expression of VGCCs, including calmodulin, Akap15, Akap9, and eIF3e, and these associations play an important role in connecting VGCCs and intracellular signaling pathways. 25 Of these proteins, ALPL is unusual because it is an ectoenzyme that hydrolyzes several substrates. Our study showed a new function for ALPL in in BMSCs; it controls Ca 2+ influx by regulating the internalization of Ca V 1.2 and Ca V 1.3. The results suggest that ectoenzymes on the cell membrane may bind channels in the cell and regulate their trafficking. Precisely how ALPL regulates L-type Ca 2+ channel trafficking is unclear. Our colocalization data indicate that there is direct contact between ALPL and L-type Ca 2+ channels. The α2δ subunit of the L-type Ca 2+ channel is responsible for regulating the trafficking of channels. 23,24 Our colocalization data indicate that ALPL may bind to the α2δ subunit to regulate L-type Ca 2+ channel trafficking. A mutation in the α2δ subunit reduced the expression of Ca V 1.2 and Ca V 1.3 at the membrane of cells. However, further studies are required to explore the detailed mechanism of L-type Ca 2+ channel trafficking. Conditional binding of ALPL to Ca V 1.2 and Ca V 1.3 suggests that the composition of the Ca V 1.2 and Ca V 1.3 protein complex with ALPL may play an important role in regulating channel trafficking. On the other hand, our previous study showed that ALPL deficiency in BMSCs enhanced ATP release and reduced ATP hydrolysis. 3 ATP, as the energy source used in channel trafficking, may also play a critical role in L-type Ca 2+ channel internalization in ALPL deficiency conditions. Skeletal defects in HPP, include rickets, osteomalacia, fractures, bone pain, and various dental defects. 33,34 To understand the physiological role of ALPL and evaluate the potential treatments, several lines of ALPL knockout mice were generated. 35,36 Homozygous mice show severe bone disease, but they often die before puberty. 37 However, here, we found that ALPL deficiency in BMSCs caused decreased osteogenic differentiation and increased adipogenic differentiation. The alpl +/− mouse model phenocopies adult patients with HPP, and these mice showed inhibited bone formation but increased adipose tissue in the bone matrix. Moreover, bone formation was inhibited when we generated alpl conditional knockout mice of BMSCs, which was consistent with recent reports. 37 Further, other recent data have shown that increased marrow adipose tissue is correlated with dysfunction of bone and hematopoietic regeneration. 38 We also found that bone formation was inhibited but adipose tissue was increased in the bone matrix of alpl conditional knockout mice, which suggests that ALPL regulates the osteogenic/adipogenic differentiation of BMSCs and causes osteoporosis in patients with HPP. Our findings regarding the involvement of ALPL in calcium homeostasis revealed a molecular mechanism underlying the BMSC balance between osteogenic and adipogenic differentiation. The change in Ca 2+ influx in BMSCs following H 2 S exposure regulates osteogenic differentiation through the PCK/Erk/Wnt pathway. 39 Here, we demonstrated that the Akt/GSK3β/Wnt/ β-catenin pathway was downstream of ALPL-mediated regulation of Ca 2+ influx. The Akt/GSK3β/Wnt/β-catenin pathway further balanced the osteogenic and adipogenic differentiation of BMSCs and bone formation. In our study, we found that ALPL was required to maintain intracellular Ca 2+ influx by regulating internalization of the L-type Ca 2+ channel via binding to the α2δ subunits in BMSCs. Altered intracellular Ca 2+ influx caused by the ALPL deficiency resulted in an osteoporotic phenotype due to downregulated osteogenic differentiation and upregulated adipogenic differentiation in both human and mouse BMSCs. Inhibition of calcium channel internalization by ionomycin increased calcium influx and enhanced bone formation in alpl +/− mice and Prrx1alpl −/− mice, suggesting that targeting calcium channel internalization is a potential treatment strategy for adult patients with HPP.
Isolation of human bone marrow mesenchymal stem cells Two HPP patients aged 10 (male) and 2.5 (female) years were treated by the Affiliated Hospital of Fourth Military Medical University for osteodynia and missing teeth. Healthy human BM samples were collected from five teenagers aged 10-13 years (male) who underwent alveolar bone cleft repair by autoilium transplantation. The cells were purified from the BM using a Percoll density gradient centrifugation method, and then they were cultured in α-MEM supplemented with 10% FBS (Gibco BRL), 2 mmol·L −1 L-glutamine (Invitrogen), 100 U·mL −1 penicillin, and 100 mg·mL −1 streptomycin (Invitrogen) at 37°C in 5% CO 2 . 3 BMSCs in their third passage were used in experiments.
Calcium imaging Calcium imaging was performed using confocal laser microscopy (Zeiss, Oberkochen FV1000, Germany). The intracellular Ca 2+ level ([Ca 2+ ] i ) was determined by Fluo-3 fluorescence intensity, as described previously. 40 Briefly, BMSCs were cultured in 12-well plates and were incubated with 5 μmol·L −1 Fluo-3/AM dye (Invitrogen, Life Technology, Carlsbad, CA, USA) for 30 min in α-MEM at 37°C. BMSCs were again washed three times with calibrated EGTA/Ca 2+ solutions. KCl (30 mmol·L −1 ) or TG (20 μmol·L −1 , Sigma-Aldrich, St. Louis, MO, USA) was added to test which type of calcium channel was affected. Images were collected every 4 s at 2 Hz with excitation at 488 nm and emission at 530 nm. Data are presented as the Fluo-3 fluorescence intensity increase ratio: R = ΔF/F 0 , where ΔF = F -F 0 . F is the fluorescence value detected, and F 0 is the minimum fluorescence value.
Confocal microscopy Confocal images were acquired with a Zeiss Oberkochen FV1000 confocal laser scanning microscope using a ×60 oil immersion objective. BMSCs were fixed with 3.7% paraformaldehyde in distilled water at 4°C for 10 min and then were incubated overnight with primary antibodies, which were followed by incubation with secondary antibodies for 1 h. The nuclei were stained with 1 μg·mL −1 Hoechst 33342. The plasma membrane was stained with 5 μg·mL −1 of the membrane marker CellMask™ Deep Red Plasma Membrane Stain (Thermo Fisher Scientific, MA, USA). Images were acquired using an argon laser (excitation, 488 nm; emission, BP505-530 nm emission filter) for FITC-labeled Ca V 1.2 or Ca V 1.3, a UV laser for excitation and a BP385-470 nm emission filter for Hoechst 33342, and a He-Ne laser (excitation, 543 nm; emission filter, LP650 nm) for Cy3-labeled ALPL. BMSCs (1 × 10 5 ) were plated onto coverslips, and the next day cells were treated with 10 μmol·L −1 ATP, 10 μmol·L −1 ppi, or 1 μg·mL −1 ionomycin for 1 h before immunofluorescence staining for Ca V 1.2 or Ca V 1.3. Plasma membrane localization of Ca V 1.2 or Ca V 1.3 in BMSCs, as visualized by staining with anti-Ca V 1.2 or anti-Ca V 1.3 antibodies, was recorded for more than ten cells. Colocalization of the L-type Ca 2+ channel (Ca V 1.2 or Ca V 1.3) and ALPL was also observed using a laser scanning confocal microscope, and images were obtained using FV10-ASW Viewer 4.2 (Zeiss, Oberkochen FV1000, Germany).
To record the time-course change of internalization of Ca V 1.2, a plasmid encoding DsRed-Cav1.2 was generated and transfected into the BMSCs. The plasmid was constructed by introducing a DsRed segment into the Ca V 1.2 plasmid (Addgene plasmid #26572) according to the instructions of a ClonExpress® II One ALP regulates Ca2+ channel in BMSCs B Li et al.
Step Cloning kit (Vazyme, Nanjing, China). Dio (Thermo Fisher Scientific, MA, USA) was used to label the cell membrane, and DAPI (Thermo Fisher Scientific) was used to label the nucleus. The average colocalization intensity was determined by selecting an ROI corresponding to the cell's footprint in the first image and measuring the average intensity in that region over the entire time course. The ROI was visually selected in a region of the Diolabeled plasma membrane. Cells that were outside of this ROI were excluded from analysis. The amount of time channels spent at the membrane was measured for more than 15 ROIs for at least five cells per condition. We recorded the time-dependent red fluorescence intensity of these regions. Quantitative data are presented as the fluorescence intensity increase ratio: R = F/F 0 . F is the fluorescence value detected, and F 0 is the first detected fluorescence value. Quantification of the fluorescence density of ROIs at 0 s, 300 s, and 600 s was analyzed by NIH ImageJ software.

Transfection
For transfecting experiments, BMSCs (0.5 × 10 6 ) were seeded on a six-well culture plate and then were transfected with Ca V 1.2 siRNA or Ca V 1.3 siRNA (Santa Cruz, Dallas, TX, USA) using X-tremeGENE siRNA Transfection Reagent (Roche, Basel, Switzerland) according to the manufacturer's instructions.
To downregulate or overexpress ALPL in BMSCs, we first produced lentiviruses carrying shALPL or ALPL cDNA. The lentiviral vector and the ViraPower Packaging Mix were cotransfected into 293T cells to produce a lentiviral stock according to the protocol provided by the manufacturer. Virus-containing supernatants were harvested 48 h after transfection, and then they were pooled and filtered through 0.45-µm filters. Cells were treated with a lentivirus at a multiplicity of infection of 100 at 37°C and 5% CO 2 . The plates were swirled every 15 min, and fresh medium was added after 1 h of incubation. The cells were exposed to lentivirus for 48 h, which was followed by protein extraction for western immunoblotting or differentiation induction.
MicroCT analysis Femurs were harvested and analyzed using a desktop microCT system (eXplore Locus SP, GE Healthcare, USA). The scanner was set at a voltage of 80 kVp, a current of 80 μA and a resolution of 8 μm per pixel. Cross-sectional images of middiaphysis femurs were used to perform three-dimensional histomorphometric analysis of trabecular bone. Cross-sectional volumetric BMD was measured for the right femur middiaphysis with a density phantom. Using three-dimensional images, an ROI in the secondary spongiosa was manually drawn near the endocortical surface. Bone volume relative to tissue volume (BV/TV) and Tb.N were assessed as cancellous bone morphometric parameters.

Histology
To assess the areas of trabecular bone and bone marrow, femurs and tibias were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and then were decalcified with 5% EDTA (pH 7.4), which was followed by paraffin embedding. The 6 μm paraffin sections were stained with hematoxylin and eosin (H&E) and were analyzed using NIH ImageJ software. To label the matrix mineralization, the mice were given intraperitoneal injections of calcein (Sigma-Aldrich, USA, 20 mg·kg −1 body weight) at day 10 and day 3 before sacrifice. Bone dynamic histomorphometric analyses for MAR were performed according to the standardized nomenclature for bone histomorphometry using fluorescence microscopy (Leica DM 6000B, German).
In vivo oil red O staining To assess adipose tissue in trabecular areas, femurs were fixed in 4% paraformaldehyde and were decalcified with 5% EDTA (pH 7.4), which was followed by cryosectioning. Sections were stained with oil red O, and positive areas were quantified under microscopy and are shown as a percentage of the total area. Briefly, sections were washed with 60% isopropanol and then were incubated in fresh oil red O staining solution for 15 min at room temperature before being counterstained with hematoxylin. All reagents for oil red O staining were purchased from Sigma-Aldrich.
Ionomycin treatment Ionomycin (Alomone, Jerusalem, Israel) was dissolved in DMSO. For in vivo treatment, ionomycin was intraperitoneally administered to 12-week-old alpl +/− mice and alpl −/− CKO mice at a dose of 1 mg·kg −1 per day for 28 days. The control mice were treated with only the vehicle. After ionomycin treatment, all groups of mice were healthy.

Statistics
All experimental group sizes were chosen to ensure adequate statistical power despite the highly variable nature of the studies performed. No animals were excluded, and animals were randomly assigned groups for the studies. Experiments were not performed in a blinded fashion. Data were assessed for normal distribution and similar variance between groups. Comparisons between two groups were performed using independent unpaired two-tailed Student's t tests, and comparisons between more than two groups were analyzed using one-way ANOVA with the Bonferroni adjustment. P values of less than 0.05 were considered statistically significant.