Panoply™ Human LRP1 Over-expressing Stable Cell Line

Single-nucleotide polymorphisms in the LRP1 gene coding sequence are associated with low bone mass, and cell culture studies have demonstrated a role for LRP1 in osteoblast proliferation and osteoblast-mediated osteoclastogenesis. However, the role of LRP1 in bone homeostasis in vivo has not been explored. Here, researchers used a C57BL/6J mouse model in a Ctsk-Cre;Lrp1^f/f background to investigate the osteoclast-specific effects of LRP1 in bone homeostasis. These mice exhibited a significant decrease in trabecular bone mass and a marked increase in osteoclast number, while osteoblast activity was unaffected or slightly increased. Cortical bone parameters were largely unchanged. Upon RANKL treatment, Lrp1-deficient bone marrow mononuclear cells differentiated more efficiently into osteoclasts and displayed enhanced p65 NFκB and p38 signaling. Consistently, Lrp1-overexpressing Raw264.7 cells exhibited desensitization to RANKL-induced p38 and p65 activation and osteoclastogenesis. Furthermore, RANKL treatment led to a dramatic decrease in LRP1 protein and RNA levels in BMMs. Together, these data indicate that osteoclast-expressed LRP1 is a key regulator of bone mass, inhibiting both the NFκB and p38 pathways and reducing the efficiency of RANKL-induced osteoclastogenesis.

Because Lrp1 deficiency promotes osteoclast formation, and Lrp1 expression decreases with osteoclastogenesis, the researchers examined the effects of increased Lrp1 on osteoclastogenesis. Following RANKL treatment, control Raw264.7 cells exhibited robust osteoclastogenesis, peaking on day 5. However, the osteoclastogenic capacity of LRP1-overexpressing Raw264.7 cells was much weaker, and this was not improved by prolonged RANKL treatment (Figure 1A). Furthermore, LRP1-overexpressing Raw264.7 cells were less sensitive to RANKL-induced NFκB and p38 activation but remained responsive to RANKL when activating the AKT, JNK, and ERK pathways (Figure 6B). By qRT-PCR, the researchers found that expression of NFκB and/or p38 downstream target genes (such as Ctsk, Ocstamp, and Oscar) in LRP1-overexpressing Raw264.7 cells was significantly lower after RANKL treatment than in control cells (Figure 1C). In the absence of RANKL, the expression of Ctsk and Ocstamp was also significantly reduced in LRP1-overexpressing Raw264.7 cells (Figure 1C). Nfatc1 and Mitf expression was also reduced in LRP1-overexpressing Raw264.7 cells, but to a lesser extent than that of Ctsk and others. However, Csf1r, Tnfrsf11a, and Tnfr1 were unaffected or only slightly affected. These data support the idea that Lrp1 acquisition may inhibit RANKL-induced osteoclastogenesis by attenuating p38 and NFκB signaling in OC precursors, rather than by modulating the expression levels of these key osteoclastogenic receptors.

Figure 1. Lrp1 overexpression desensitized RANKL-induced osteoclastogenesis in the RAW264.7 cells. (Lu D, et al., 2018)