article

Loss of Phosphatidylinositol-4-Phosphate 5-Kinase Type-1 Gamma (Pip5k1c) in Mesenchymal Stem Cells Leads to Osteopenia by Impairing Bone Remodeling

Introduction Bone is a dynamic tissue that is constantly changing and is made up of two processes: bone resorption and bone creation. Osteoclasts govern bone resorption by removing the aged or diseased bone and producing resorption cavities. Osteoblasts enter the cavities and secrete chemicals that fill the pit and generate new bone. Bone production and resorption are geographically and temporally related processes that must be properly managed. Osteopenia and osteopetrosis are metabolic bone disorders caused by an imbalance in bone resorption and production. The study discovered that Pip5k1c deficiency reduces osteoclast production, osteoid, and mineral deposition, and so does bone formation. Phosphatidylinositol-4-phosphate 5-kinase type-1 gamma (Pip5k1c) is a lipid kinase that regulates receptor-mediated calcium signaling in a variety of organs. Using Prx1-Cre transgenic mice, eliminating Pip5k1c expression in mesenchymal stem cells did not impede intramembranous and endochondral ossification during skeletal development, but it does cause osteopenia in adult mice but not in fast-developing young animals. Furthermore, Pip5k1c deficiency suppresses osteoblastic development while promoting adipogenic differentiation of bone marrow stromal cells. Pip5k1c deficiency also reduces cytoplasmic calcium influx and inactivates the calcium/calmodulin-dependent protein kinase, which regulates Runx2 levels via modifying its stability and consequent osteoblast and bone production. Furthermore, Pip5k1c deficiency decreases levels of the nuclear factor-B receptor activator, but not osteoprotegerin, its decoy receptor, in osteoblasts in bone and serum. Finally, we discovered that Pip5k1c deficiency inhibits bone marrow stromal cell capacity to promote osteoclast formation of bone marrow monocytes and decreases the osteoclast precursor population in bone marrow, resulting in decreased osteoclast production and bone resorption.

Methods The exon two of the mouse Pip5k1c gene is flanked by two loxP sites and followed by a FRT site-flanked neomycin cassette before exon 3. Homologous recombina- tion using the targeted Pip5k1c construct was performed in embryonic stem cells derived from 129 mouse substrains, and neomycin-resistant clones were selected. Recombinant em- bryonic stem cell clones were injected into C57BL/6 mouse embryos to generate chimeric mice. Progenies of the chimeric mice containing the targeted Pip5k1c allele were crossed with Flp recombinase transgenic mice to generate Pip5k1c loxP/wt; Flp mice, in which the neomycin cassette was removed. These animals were backcrossed with WT C57BL/6 mice six times to remove the recombinase and dilute the 129s genomic back- ground. Pip5k1c loxP/wt mice were then self-crossed to generate Pip5k1c loxP/loxP mice (referred to as Pip5k1cfl/fl hereafter). To generate Prx1-Cre; Pip5k1cfl/fl mice (referred to as Pip5k1cPrx1 or cKO hereafter), Prx1-Cre transgenic mice with Cre expression in the limb and head mesenchyme driven by the 2.4-kb Prx1 (paired-related homeobox gene-1) gene promoter were crossed with Pip5k1cfl/fl mice. The Cre- negative Pip5k1cfl/fl littermates were used as control (referred to as WT) mice in this study. Mice were caged at the density of 4 to 6 mice/cage with free access to food and water. Room temperature was maintained between 20 to 24 C. All mice were maintained in C57BL/6 background.

Results/Data

Deleting Pi5k1c expression in limb and head MSCs had little effect on skeletal development in mice. The results of quantitative real-time RT-PCR analysis of total RNA extracted from the respective organs of E14.5 control and cKO mice revealed that the amount of Pip5k1c mRNA in the forelimb, but not in the lung, liver, heart, or brain, was significantly lower in cKO mice compared to control animals (Fig 1B). In cKO mice, alizarin red and alcian blue double staining of whole-mount skeletons revealed no significant anomalies in the cranial vault, forelimb, hindlimb, clavicle, rib cage, or sterna. These findings imply that Pip5k1c deletion has no effect on both endochondral and intramembranous ossification during early skeletal development.

Figure 1 Deletion of Pip5k1c in limb and head mesenchymal stem cells does not affect skeletal development in mice.

Adult mice with Pip5k1c deletion have poor bone mass, whereas fast developing young mice do not. It discovered that cKO mice had significantly less trabecular bone mass than their control littermates. The deletion of Pip5k1c had no discernible effect on trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), cortical thickness, cortical area, or marrow area. In 4-month-old female cKO mice, a comparable osteopenia was seen (data not shown). It should be highlighted that in 1-month-old cKO male and female mice, this osteopenic phenotype was not found (data not shown). Furthermore, CT examination of the skull vault revealed that BMD, BV/TV, Tb.N, Tb.Sp, and Tb.Th were equivalent in control and cKO mice. CT scans indicated no significant changes in the spinal vertebral body bone and alveolar bone difference between the two genotypes at 4 months of age.

Figure 2 Pip5k1cPrx1 cKO mice display low bone mass at adult stage.

In vivo, the loss of Pip5k1c substantially affects osteoblast development, osteoid and mineral synthesis, and bone formation. The blood procollagen type 1 amino-terminal propeptide (P1NP), an indication of in vivo osteoblast activity and bone production, was considerably lower in 4-month-old cKO mice compared to control littermates, according to ELISA results. In vitro, Pip5k1c deletion reduces colony forming unit-fibroblast and colony forming unit-osteoblast and osteoblast differentiation while increasing adipogenic differentiation of bone marrow stromal cells. Adipocyte production and gene expression were both higher in the cKO group compared to the control group. In cKO mice, there was a significant increase in adiposity in the BM of the distal tibiae compared to control littermates. Pip5k1c deficiency reduces Runx2 protein levels in BMSC through decreasing cytoplasmic Ca2+ levels and CaMK activation. Both Rankl and osteoprotegerin (Opg) are important regulators of osteoclast development and differentiation. In vitro osteoclast formation experiments revealed that cKO mice's primary bone marrow monocytes (BMMs) produced fewer multinucleated TRAP positive osteoclasts than control littermates. The number of TRAP positive multinucleated cells (MNCs, defined as having three or more nuclei per cell) was reduced by nearly half in cKO BMM cultures compared to control BMM cultures. Cells in BM with CD3-CD45R-CD11b-/low CD117+ CD115hi had the greatest capacity to develop into osteoclasts in vitro. BMSCs from cKO mice are ineffective in supporting osteoclast development. BMSCs from cKO mice are ineffective in supporting osteoclast development.

Discussion The study found that a lack of Pip5k1c in MSC promotes osteopenia in adults but not in fast developing young mice. Notably, Pip5k1c deletion in MSC did not impede intramembranous or endochondral ossification during skeletal development. We show that the deletion of Pip5k1c in MSC produces low-turnover osteopenia in mice, with osteoblast and bone production deficits being larger than those of osteoclast formation and bone resorption. It demonstrates that Pip5k1c expression in MSC is required for bone osteoclast production. Pip5k1c deficiency also inhibits Runx2 protein expression in BMSC through inactivating CaMK2. Because Pip5k1c is not deleted in osteoclasts or their precursors, the findings imply that Pip5k1c deletion in MSC alters the BM microenvironment. Critical analysis limitations The study defines a critical role of Pip5k1c in the control of bone mass, and its ablation in MSC causes low-turnover osteopenia in mice through distinct mechanisms and provides a potential therapeutic target for osteoporosis. The objective of this research is related to the bone resorption process which takes approximately 12-16 weeks. Hence, it is considered a long experiment to observe and analyze the data with several complicated laboratory types of equipment.

References

Yan, Q., Gao, H., Yao, Q., King, K., Xiao, G. (2022). Loss of Phosphotidyllinositol-4-phosphate 5 Kinase Type-1 Gamma (Pip5k1c) in Mesenchymal stem Cells Leads to Osteopenia by Impairing Bone Remodeling. MCP Research, 298(3), 101639. https://doi.org/10.1016/j.jbc.2022.101639