BoneKEy-Osteovision | Commentary
A genetic dissection of IKKα functions
Nuclear factor-κB (NF-κB) was first identified in 1986 as a nuclear factor that bound to an enhancer element of the immunoglobulin (Ig)κ light chain gene, and was believed to be specifically expressed in B lymphocytes (). Now, NF-κB is a collective name for inducible dimeric transcription factors from the rel family found in virtually all cell types and involved in activation of an extremely large numbers of genes in response to infections, inflammation, and during carcinogenesis (). The pathway that leads to NF-κB transcriptional activation is unique. NF-κB is normally sequestered inactive in the cytoplasm through association with an endogenous inhibitor protein IκB (inhibitor of NF-κB) family. Exposure of cells to a variety of stimuli leads to the rapid phosphorylation, ubiquitination, and proteolytic degradation of IκB, which frees NF-κB and allows its translocation to the nucleus where it regulates gene transcription. The multisubunit IκB kinase (IKK) responsible for IκB phosphorylation is the point of convergence for most NF-κB-activating stimuli. IKK contains two catalytic subunits, IKKα and IKKβ, both of which are able to phosphorylate IκB correctly. Elegant gene knockout experiments have demonstrated that despite their structural and biochemical similarities, IKKα and IKKβ are indeed functional distinct. IKKβ is essential for NF-κB activation in response to proinflammatory stimuli and for prevention of TNF-α induced apoptosis; furthermore, it activates NF-κB through the canonical pathway described above (). Conversely, IKKα is not required for such responses, but it seems to have unique roles that are either independent of NF-κB activation (), or dependent on increased NF-κB transcriptional activity through a pathway that does not involve IκB (). In particular, universal deletion of the IKKα gene as result of homologous has been reported to cause lethality at birth and severe skin abnormalities, but no apparent impairment of activation NF-κB (). Taken together, these surprising results generated an interesting question: is there any IKKα biological function that is mediated by NF-κB activation through the canonical pathway?
The study recently published by Michael Karin and colleagues in Cell () is the first report to describe an IKKα biological function that depends on degradation of IκB and, consequently, NF-κB activation. Mice had been previously generated by the same group that carried a “knockin IkkαAA” allele, in which serines whose phosphorylation is required for kinase activity were replaced with alanines. The homozygous IkkαAA mutant mice had been reported to be viable, fertile, healthy and phenotypically normal, but with a specific defect in B lymphocyte maturation due to impairment of NF-κB activation through a pathway not involving IκB (). In this paper, Cao et al. now report the novel finding that homozygous IkkαAA females display a severe lactation defect due to defective proliferation of the lobulo-alveolar tree during pregnancy. Interestingly, whole mount analysis of mammary glands demonstrated that homozygous virgin females completed normal ductal development; this result strongly suggested that the mutant mice had a selective defect of lobulo-alveolar development exclusively during pregnancy and lactation. Furthermore, the authors convincingly show that the defect is autonomous to the mammary epithelium, and is due to severely reduced cell proliferation, rather than to increased cell death. With a series of clever genetic manipulations, Cao et al. have been able to dissect genetically the pathway that involves IKKα in mammary gland development, and this has led to the identification of the first function of IKKα that depends on NF-κB activation through degradation of IκB. And they have gone further with the study, by convincingly proving with the use of genetic models that RANKL-RANK- and cyclin D1 are critical component of the pathway. Interestingly enough, TNF-α - one of the classical stimuli upstream of IKKβ activity - seems not to be involved in the activation of this signaling pathway.
The results reported by Cao et al. are exciting and important for numerous reasons. They define for the first time a biological role of IKKα that depends on Iκb degradation and cannot be replaced by IKKβ. In addition, they demonstrate different molecular requirements for the NF-κB-dependent and -independent functions of IKKα, respectively. In this regard, the homozygous IkkαAA mice show impairment of B lymphocyte maturation and mammary epithelium proliferation (NF-κ B dependent functions), but they do not display the severe skin abnormalities (NF-κB independent functions) that are typical of the Ikkα-/- and lead to perinatal death. Furthermore, the data presented by Cao et al. clearly show that IKKα and IKKβ can be differentially deployed by different members of the TNF and TNFR family to induce IκB phosphorylation and NF-κB activation. Lastly, the study provides a link between the RANKL-IKKα-IκB-NF-κB signaling pathway and cyclin D1 expression (known to be involved in development of breast tumors ()) in mammary gland development.
Further investigations will be needed in order to identify a missing piece of the puzzle, i.e. the molecular mechanism that is responsible for the different responsiveness of IKKα and IKKβ to different stimuli.
IkkαAA mutation causes the same defect in mammary gland development as the ablation of either RANK () or RANKL (), i.e. in all the three cases the defect is restricted to growth of the lobulo-alveolar network just during pregnancy. However, differently from Rank -/- and Rankl -/- animals, IkkαAA mutant mice do not develop osteopetrosis, further proving the selective and highly specific role of IKKα in activating NF-κB transcriptional activity during mammary gland development. The RANK-RANKL system plays a critical role in controlling osteoclast activation, and recent data support the notion that activation of NF-κB pathway is an integral component of RANKL induced osteoclast differentiation (). Interestingly, the study by Cao et al. suggests that phosphorylation of IKKα is not required for this RANKL-dependent function. RANKL mRNA production has been shown to be upregulated by PTHrP in the mammary epithelium (); furthermore, the lack of PTHrP causes a severe impairment in mammary gland development (). Is RANKL-NF-κB signaling pathway a major downstream effector of PTHrP in mammary epithelium?
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