Loss of chaperone-mediated autophagy is associated with low vertebral cancellous bone mass.

Chaperone-mediated autophagy (CMA) is a protein degradation pathway that eliminates soluble cytoplasmic proteins that are damaged, incorrectly folded, or targeted for selective proteome remodeling. However, the role of CMA in skeletal homeostasis under physiological and pathophysiological conditions is unknown. To address the role of CMA for skeletal homeostasis, we deleted an essential component of the CMA process, namely Lamp2a, from the mouse genome. CRISPR-Cas9-based genome editing led to the deletion of both Lamp2a and Lamp2c, another Lamp2 isoform, producing Lamp2AC global knockout (L2ACgKO) mice. At 5 weeks of age female L2ACgKO mice had lower vertebral cancellous bone mass compared to wild-type (WT) controls, whereas there was no difference between genotypes in male mice at this age. The low bone mass of L2ACgKO mice was associated with elevated RANKL expression and the osteoclast marker genes Trap and Cathepsin K. At 18 weeks of age, both male and female L2ACgKO mice had lower vertebral cancellous bone mass compared to WT controls. The low bone mass of L2ACgKO mice was associated with increased osteoclastogenesis and decreased mineral deposition in cultured cells. Consistent with these findings, specific knockdown of Lamp2a in an osteoblastic cell line increased RANKL expression and decreased mineral deposition. Moreover, similar to what has been observed in other cell types, macroautophagy and proteasomal degradation were upregulated in CMA-deficient osteoblasts in culture. Thus, an increase in other protein degradation pathways may partially compensate for the loss of CMA in osteoblasts. Taken together, our results suggest that CMA plays a role in vertebral cancellous bone mass accrual in young adult mice and that this may be due to an inhibitory role of CMA on osteoclastogenesis or a positive role of CMA in osteoblast formation or function.

Deletion of a putative promoter-proximal Tnfsf11 regulatory region in mice does not alter bone mass or Tnfsf11 expression in vivo.

The cytokine RANKL is essential for osteoclast formation during physiological and pathological bone resorption. RANKL also contributes to lymphocyte production, development of lymph nodes and mammary glands, as well as other biological activities. Transcriptional control of the Tnfsf11 gene, which encodes RANKL, is complex and involves distant regulatory regions. Nevertheless, cell culture studies suggest that an enhancer region near the transcription start site is involved in the control of Tnfsf11 expression by hormones such as 1,25-(OH)2 vitamin D3 and parathyroid hormone, as well as the sympathetic nervous system. To address the significance of this region in vivo, we deleted the sequence between -510 to -1413 bp, relative to Tnfsf11 exon 1, from mice using CRISPR-based gene editing. MicroCT analysis of the femur and fourth lumbar vertebra of enhancer knockout mice showed no differences in bone mass compared to wild type littermates at 5 weeks and 6 months of age, suggesting no changes in osteoclast formation. RNA extracted from the tibia, fifth lumbar vertebra, thymus, and spleen at 6 months of age also showed no reduction in Tnfsf11 mRNA abundance between these groups. However, maximal stimulation of Tnfsf11 mRNA abundance in cultured stromal cells by PTH was reduced approximately 40% by enhancer deletion, while stimulation by 1,25-(OH)2 vitamin D3 was unaffected. The abundance of B and T lymphocytes in the bone marrow did not differ between genotypes. These results demonstrate that the region between -510 and -1413 does not contribute to Tnfsf11 expression, osteoclast support, or lymphocyte production in mice under normal physiological conditions but may be involved in situations of elevated parathyroid hormone.

Osteocyte RANKL is required for cortical bone loss with age and is induced by senescence.

In aging mice, osteoclast number increases in cortical bone but declines in trabecular bone, suggesting that different mechanisms underlie age-associated bone loss in these 2 compartments. Osteocytes produce the osteoclastogenic cytokine RANKL, encoded by Tnfsf11. Tnfsf11 mRNA increases in cortical bone of aged mice, suggesting a mechanism underlying the bone loss. To address this possibility, we aged mice lacking RANKL in osteocytes. Whereas control mice lost cortical bone between 8 and 24 months of age, mice lacking RANKL in osteocytes gained cortical bone during this period. Mice of both genotypes lost trabecular bone with age. Osteoclasts increased with age in cortical bone of control mice but not in RANKL conditional knockout mice. Induction of cellular senescence increased RANKL production in murine and human cell culture models, suggesting an explanation for elevated RANKL levels with age. Overexpression of the senescence-associated transcription factor Gata4 stimulated Tnfsf11 expression in cultured murine osteoblastic cells. Finally, elimination of senescent cells from aged mice using senolytic compounds reduced Tnfsf11 mRNA in cortical bone. Our results demonstrate the requirement of osteocyte-derived RANKL for age-associated cortical bone loss and suggest that increased Tnfsf11 expression with age results from accumulation of senescent cells in cortical bone.

Local Production of Osteoprotegerin by Osteoblasts Suppresses Bone Resorption.

Osteoprotegerin (OPG) inhibits the ability of receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) to stimulate the differentiation, activity, and survival of bone-resorbing osteoclasts. Genetic studies in mice show that osteocytes are an important source of RANKL, but the cellular sources of OPG are unclear. We use conditional deletion of Tnfrsf11b, which encodes OPG, from different cell populations to identify functionally relevant sources of OPG in mice. Deletion from B lymphocytes and osteocytes, two cell types commonly thought to supply OPG, has little or no impact on bone mass. By contrast, deletion of Tnfrsf11b from osteoblasts increases bone resorption and reduces bone mass to an extent similar to germline deletion, demonstrating that osteoblasts are an essential source of OPG. These results suggest that, in addition to producing new bone matrix, osteoblasts also play an active role in terminating the resorption phase of the bone remodeling cycle by suppressing RANKL activity.

Effective CRISPR interference of an endogenous gene via a single transgene in mice.

Drawbacks of conditional gene deletion in mice include the need for extensive breeding and, often, a lack of cell type-specificity. CRISPR interference (CRISPRi) is an alternative approach for loss-of-function studies that inhibits expression by guiding a transcriptional repressor to the transcription start-site of target genes. However, there has been limited exploration of CRISPRi in mice. We tested the effectiveness of a single CRISPRi transgene broadly expressing a single guide RNA and a catalytically dead Cas9 fused to the KRAB repressor domain to suppress a well-characterized target gene, Tnfsf11. The phenotype of CRISPRi transgenic mice was compared to mice with germline deletion of Tnfsf11, which are osteopetrotic and do not form lymph nodes. High transgene expression mimicked gene deletion, with failure of lymph node development and classic signs of osteopetrosis such as high bone mass and failure of tooth eruption. Mice with low transgene expression were normal and mice with medium expression displayed an intermediate phenotype. Transgene expression in tissues from these mice correlated inversely with Tnfsf11 mRNA levels. These results demonstrate that a single CRISPRi transgene can effectively suppress a target gene in mice and suggest that this approach may be useful for cell type-specific loss-of-function studies.