In this study, we set out to examine the role of alternative NF-κB signaling in the osteolineage by targeting a key upstream kinase, IKKα, using Osx-cre. Previously, we found that forced activation of this pathway using a constitutively active allele of NIK with the same Osx-cre driver enhanced both basal and stimulated bone formation 14. Here, male cKO mice showed no differences in bone mass up to 18 months of age, and mechanical loading by tibial compression failed to generate any differences in bone formation. Female mice also had normal bone mass at 4 months of age. Thus, under basal and non-inflammatory loading conditions, IKKα does not seem to have a clear role, either positive or negative, in bone formation. One limitation of our bone analysis is that we did not follow females over time, despite our finding that global loss of alternative NF-κB components NIK and RelB has greater effect in females. However, in those models, the differences are clear by 10 weeks of age. It is possible that IKKα may impact bone formation in the context of strong inflammatory stimuli such as inflammatory arthritis models, which were not examined here.
Although Osx-Cre efficiently drives recombination in osteoblasts, not all phenotypes identified can be attributed to effects in the osteolineage. The Osx-Cre allele has established recombination activity in many extraskeletal tissues including synovium 25, intestinal epithelium 26,27,, and kidney 28, as well as in some hematopoietic stem and progenitor cells 29. In tumor bearing mice, Osterix, as well as the Osx-Cre allele, is also expressed in a subset of cancer-associated fibroblasts with a dual fibroblast/osteogenic signature 29. Recently, we found subcutaneous sarcomas, but not bone tumors, in mice expressing a transgene driven by Osterix-cre 30. Therefore, given the striking loss of fat in aging cKO mice in the absence of any changes in bone, we considered the possibility that recombination outside of bone was responsible for the phenotype. PCR of genomic DNA from peripheral fat depots in middle aged mice showed some recombination of the IKKα allele, albeit less robust than in bone. Examination of inguinal fat pads from reporter mice showed robust TdT expression only in adipocytes from aged mice. Despite identification of TdT in several subsets of CD45+ cells in a previous study using the same line of Osx-Cre;TdT mice 29, we did not identify any such cells in the inguinal fat sections at any age. We therefore concluded that Osx-Cre can drive recombination in adipocytes, and this cell autonomous effect is the most likely cause of the cKO fat phenotype. Future experiments using an adipose-specific cre driver are required to confirm this and would be useful to determine the mechanism by which IKKα impacts adipocyte metabolism.
In addition to the unexpected finding of a fat phenotype driven by Osx-Cre, we also did not anticipate the strong effect of age on activation of this Cre. Previous studies utilizing this Cre driver primarily utilize mice under 6 months of age, and the few studies with mice at or beyond 1 year of age did not describe, or specifically look for, Cre expression outside of bone 31-33. More comprehensive analysis of conditional mouse models with sensitive reporters like TdTomato is likely to uncover other so-called off-target effects that arise with aging in many Cre lines.
Most studies of the role of NF-κB in adipocytes have focused on IKKβ, an apex kinase in the canonical pathway. Loss of IKKβ in mature adipocytes using Adiponectin-Cre or Adipoq-Cre did not affect body weight in normal or HFD conditions 34,35. However, although fat mass was decreased in these models, the mice were insulin resistant on HFD. In contrast, with conditional loss of IKKβ in adipocyte progenitors, lower fat mass on HFD was accompanied by improved glucose homeostasis 36. Constitutive activation of IKKβ in adipocytes prevented diet-induced obesity and improved glucose tolerance 37. Interestingly, IKKβ in adipocytes may act via direct phosphorylation of targets such as β-catenin and BAD 34,38, rather than via activation of canonical NF-κB. Thus, the role of IKKβ in the adipocyte lineage is complex. Furthermore, the effects of aging have not been studied in any of these models.
Data on the role of IKKα or other components of alternative NF-κB in metabolism is even more limited, with effects described in pancreatic islets, hepatocytes, and skeletal muscle 9. However, there has been an absence of conditional knockout studies for the pathway in peripheral adipocytes. Our cKO model, although not initially designed to target fat or metabolism, nevertheless highlights the importance of IKKα in peripheral fat, both with aging and diet-induced obesity. Whether IKKα in adipocytes acts through alternative NF-κB signaling, via other downstream kinase targets, or in a kinase-independent fashion 39, remains to be established.
Since this study was undertaken to examine the role of IKKα in bone, studies of fat and metabolism were not initially planned. By the time the fat phenotype in aging males was discovered, it was not practical to undertake a similar study in females. Therefore, we decided to investigate whether differences in fat could be accelerated using HFD in both sexes. Interestingly, although both male and female cKO mice had less fat than CON after 8 weeks on the obesogenic diet, the difference in overall body weight was more pronounced in females. Thus, like osteoclasts 13, adipocytes may have differential sensitivity to the alternative NF-κB pathway by sex.
In sum, using an Osx-Cre driven conditional knockout approach, we found no clear role for IKKα in the osteolineage in either basal or mechanically stimulated conditions, but rather an intriguing role for IKKα in fat accumulation with age or diet-induced obesity. Although further experiments specifically targeting adipocytes are needed, the results shown here suggest that inhibition of IKKα, or potentially other alternative NF-κB pathway components, may reduce fat deposition with age and improve glucose metabolism. With increasing recognition of bone as an endocrine organ, it is tempting to conclude that phenotypes arising from conditional alleles driven by Osx-Cre are due to the osteolineage. However, our finding of age-related changes in Osx-Cre expression in peripheral fat indicates that expression outside of bone should be considered when metabolic phenotypes are identified in aged animals.