Characterization of Cre recombinase mouse lines enabling cell type-specific targeting of postnatal intervertebral discs.

Cre/loxP technology is an important tool for studying cell type-specific gene functions. Cre recombinase mouse lines, including Agc1-CreERT2 , Col2a1-Cre; Col2a1-CreERT2 , Shh-Cre, Shh-CreERT2 , and Osx-Cre, have been proven to be valuable tools to elucidate the biology of long bones, yet the information for their activity in postnatal intervertebral disc (IVD) tissues was very limited. In this study, we used R26-mTmG fluorescent reporter to systematically analyze cell specificity and targeting efficiency of these six mouse lines in IVD tissues at postnatal growing and adult stages. We found that Agc1-CreERT2 is effective to direct recombination in all components of IVDs, including annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplate (CEP), upon tamoxifen induction at either 2 weeks or 2 months of ages. Moreover, Col2a1-Cre targets most of the cells in IVDs, except for some cells in the outer AF (OAF) and NP. In contrast, the activity of Col2a1-CreERT2 is mainly limited to the IAF of IVD tissues at either stage of tamoxifen injection. Similarly, Shh-Cre directs recombination specifically in all NP cells, whereas Shh-CreERT2 is active only in a few NP cells when tamoxifen is administered at either stage. Finally, Osx-Cre targets cells in the CEP, but not in the NP or AF of IVDs tissues at these two stages. Thus, our data demonstrated that all these Cre lines can direct recombination in IVD tissues at postnatal stages with different cell type specificity and/or targeting efficiency, and can, therefore, serve as valuable tools to dissect cell type-specific gene functions in IVD development and homeostasis.

Cell type-specific effects of Notch signaling activation on intervertebral discs: Implications for intervertebral disc degeneration.

Intervertebral disc (IVD) degeneration is the major cause of back pain. Notch signaling is activated in annulus fibrosus (AF) and nucleus pulposus (NP) tissues of degenerated IVDs, and induced by IL1-ß and TNF-α in NP cells. However, the role of Notch activatin in the pathogenesis of IVD degeneration is largely unknown. In this study, we overexpressed the Notch1 intracellular domain (NICD1) in AF, NP, and chondrogenic ATDC5 cells via adenoviruses. Overexpression of NICD1 activated transcription of Notch signaling target genes in AF, NP, and ATDC5 cells, and caused cell type-specific effects on expression of matrix anabolic and catabolic genes. Activation of Notch signaling promoted expression of matrix catabolic genes and inhibited expression of matrix anabolic genes in both AF and ATDC5 cells, whereas its activation suppressed expression of matrix catabolic genes (including Mmp3, Mmp13, Adamts4, and Adamts5) and attenuated TNF-α and inflammatory macrophage-induced Mmp13 expression in NP cells. Consistently, sustained activation of Notch1 signaling in postnatal IVDs in mice severely disrupted growth plate and endplate cartilage tissues, but did not overly affect NP tissues. Together, these data indicated that activation of Notch signaling exerted differential and cell type-specific effects in intervertebral discs, and specific Notch signaling regulation may be considered during the treatment of IVD degeneration.

Osteocyte-intrinsic mTORC1 signaling restrains trabecular bone accrual in mice.

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signaling plays important physiological roles in bone homeostasis by regulating multiple steps of osteoblast differentiation as well as its activity. However, its potential role in osteocytes has not been explored. In this study, we deleted Raptor, a specific and essential component of mTORC1, in osteocytes using Dmp1-Cre. Deletion of Raptor in osteocytes did not affect bone development and growth, but caused compartment-specific effects on bone mass. Osteocyte-specific deletion of Raptor had no obvious effect on cortical bone compartments, but led to increased trabecular bone mass. Mechanistically, Raptor deletion resulted in decreased bone resorption without altering bone formation activity. Thus, our study revealed an unexpected role of osteocyte-intrinsic mTORC1 signaling in limiting trabecular bone mass, suggesting that osteocyte-specific inhibition of mTORC1 may be used as a novel approach to treatment of osteoporosis.

Hedgehog Signaling Controls Bone Homeostasis by Regulating Osteogenic/Adipogenic Fate of Skeletal Stem/Progenitor Cells in Mice.

Skeletal stem/progenitor cells (SSPCs) can differentiate into osteogenic or adipogenic lineage. The mechanism governing lineage allocation of SSPCs is still not completely understood. Hedgehog (Hh) signaling plays an essential role in specifying osteogenic fate of mesenchymal progenitors during embryogenesis. However, it is still unclear whether Hh signaling is required for lineage allocation of SSPCs in postnatal skeleton, and whether its dysregulation is related to age-related osteoporosis. Here, we demonstrated that Hh signaling was activated in metaphyseal SSPCs during osteogenic differentiation in the adult skeleton, and its activity decreased with aging. Inactivation of Hh signaling by genetic ablation of Smo, a key molecule in Hh signaling, in Osx-Cre-targeted SSPCs and hypertrophic chondrocytes led to decreased bone formation and increased bone marrow adiposity, two key pathological features of age-related osteoporosis. Moreover, we found that the bone-fat imbalance phenotype caused by Smo deletion mainly resulted from aberrant allocation of SSPCs toward adipogenic lineage at the expense of osteogenic differentiation, but not due to accelerated transdifferentiation of chondrocytes into adipocytes. Mechanistically, we found that Hh signaling regulated osteoblast versus adipocyte fate of SSPCs partly through upregulating Wnt signaling. Thus, our results indicate that Hh signaling regulates bone homeostasis and age-related osteoporosis by acting as a critical switch of cell fate decisions of Osx-Cre-targeted SSPCs in mice and suggest that Hh signaling may serve as a potential therapeutic target for the treatment of osteoporosis and other metabolic bone diseases. © 2021 American Society for Bone and Mineral Research (ASBMR).