IKKα is a critical coregulator of a Smad4-independent TGFβ-Smad2/3 signaling pathway that controls keratinocyte differentiation

Abstract

Cell-cycle exit and differentiation of suprabasal epidermal keratinocytes require nuclear IB kinase ␣ (IKK␣), but not its protein kinase activity. IKK␣ also is a suppressor of squamous cell carcinoma (SCC), but its mode of action remains elusive. Postulating that IKK␣ may serve as a transcriptional regulator in keratinocytes, we searched for cell-cycle-related genes that could illuminate this function. IKK␣ was found to control several Myc antagonists, including Mad1, Mad2, and Ovol1, through the association with TGF␤-regulated Smad2/3 transcription factors and is required for Smad3 recruitment to at least one of these targets. Surprisingly, Smad2/3-dependent Mad1 induction and keratinocyte differentiation are independent of Smad4, the almost universal coregulator of canonical TGF␤ signaling. IKK␣ also is needed for nuclear accumulation of activated Smad2/3 in the epidermis, and Smad2/3 are required for epidermal differentiation. We suggest that a TGF␤-Smad2/3-IKK␣ axis is a critical Smad4-independent regulator of keratinocyte proliferation and differentiation.epidermis ͉ cornification ͉ terminal differentiation A critical mediator of NF-B activation (1), IB kinase (IKK) consists of two catalytic subunits, IKK␣ and IKK␤ (2-5), and a regulatory subunit, IKK␥/NEMO (6, 7). Despite structural similarity, IKK␣ and IKK␤ have nonredundant functions, with IKK␤ being the predominant IKK (1, 8) and IKK␣ being a critical regulator of keratinocyte differentiation (9, 10). Without IKK␣, epidermal keratinocytes exhibit enhanced proliferation and failure to differentiate. Consequently, Ikk␣ Ϫ/Ϫ mice are born enshrouded in a taut and thickened, nonstratified, epidermal sheet devoid of barrier function.The mammalian epidermis is a stratified squamous epithelium in which basal keratinocytes undergo asymmetric cell divisions, giving rise to nonproliferative progeny that embark on a differentiation program as they delaminate and move upward through the spinous and granular layers before generating the cornified layer, which provides the crucial barrier function (11, 12). Without IKK␣, this process is blocked, and basal keratinocytes fail to exit the cell cycle (9, 10, 13). Isolated Ikk␣ Ϫ/Ϫ keratinocytes proliferate uncontrollably and do not respond to differentiation-inducing signals such as high Ca 2ϩ (9, 13). The reexpression of IKK␣ in Ikk␣ Ϫ/Ϫ keratinocytes induces growth arrest and allows terminal differentiation, but this function depends neither on IKK␣'s protein kinase activity nor on NF-B. Instead, it requires nuclear accumulation of IKK␣ (14).Recently, IKK␣ was identified as a tumor suppressor in squamous cell carcinoma (SCC), a type of cancer derived from squamous epithelia of the skin, oral and nasal cavities, esophagus, and other sites (15). Decreased nuclear IKK␣ expression was found in about one third of oral SCCs, mainly those that exhibit poorly differentiated phenotype and poor prognosis (16). These results strongly suggest that loss of nuclear IKK␣ contributes to malignant conversion of keratinocytes to less dif...

IKKα represses a network of inflammation and proliferation pathways and elevates c-Myc antagonists and differentiation in a dose-dependent manner in the skin

“…15 Also, it has been reported that IKKa regulates the expression of Max dimer protein 1 (Mad1) and ovo-like 1 (Ovol1), which are differentiation inducers and c-Myc antagonists. 20 Thus, we evaluated whether different levels of transgenic IKKa have different effects on EGFR activity, and on Mad1 and Ovol1 expression levels. Western blotting showed that Tg-7-IKKa and Tg-4-IKKa not only repressed EGFR activity in the skins of mice with a WT background, but also strongly repressed IKKa loss-induced EGFR activity in the skin of Ikka À/À mice in an IKKa dose-dependent manner (Figure 6a).…”

“…Thus, IKKa doses are also important for suppressing excessive mitosis of keratinocytes. On the other hand, Descargues et al 20 and Marinari et al 19 have shown that IKKa regulates the expression of Mad1 and Ovol1, c-Myc antagonists, through transforming growth factor-b/smad, in keratinocytes, skin, and skin tumors in human and mice, and that Mad1 and Ovol1 are required to induce keratinocyte differentiation and inhibit keratinocyte proliferation. In the present study, we found that transgenic IKKa substantially induced the expression of Mad1 and Ovol1 in a dose-dependent manner, and that the expression levels of Mad1 and Ovol1 were well correlated with IKKa levels and differentiation levels in the skin and keratinocytes.…”

“…However, when endogenous IKKa was absent, the epidermis of Tg-4/Ikka À/À mice expressed much higher levels of filaggrin Transforming growth factor-b and transcription regulation were previously described. 15,20 kIDF, keratinocyte-inducible differentiation factor(s). 28 Transcriptional regulation was previously described.…”

“…Several laboratories have demonstrated that IKKa expression was downregulated, or that its localization was altered in human squamous cell carcinomas of the skin, lungs, and head and neck, [16][17][18][19][20] highlighting the importance of IKKa in human malignancy development. We showed that induced IKKa deletion in keratinocytes causes epidermal hyperplasia and spontaneous skin tumors.…”

“…Moreover, studies have demonstrated that IKKa upregulates c-Myc antagonists by transforming growth factor-b and Smad pathways in coordinately regulating keratinocyte differentiation and proliferation. 19,20 Thus, IKKa acts as a sentry, monitoring the skin and, when necessary, halting keratinocyte hyperproliferation by multiple avenues.…”