Id1 was originally recognized as a negative regulator of helix-loop-helix DNA binding proteins (5
). Later on, it was shown to be involved in neurogenesis and angiogenesis of tumor xenogrants (24
). We demonstrated for the first time that Id1 was involved in mucosal infectious diseases (1
) and tied to proliferative middle ear diseases in this study.
As expected, Id1 is linked to the aggressive growth of acquired cholesteatoma through the Id1→NF-κB→cyclin D1→Ki67 signaling pathway (). First, Id1 activates the activity of NF-κB. NF-κB, in turn, activates the transcription of cyclin D1. Cyclin D1 then increases the progression of cell cycles from Go to S phase. In addition, Id1 inhibits the expression of p16Ink4a, liberating cells from cell cycle inhibition. The fact of Id1 acting through the NF-κB/cyclin D1/Ki67 signaling pathway provides an explanation as to why middle ear infection is linked to cholesteatoma. It is not clear at the moment whether physical damage and trauma to the tympanic membrane trigger the expression of Id family.
Numerous mechanisms are proposed to underlie acquired cholesteatoma. All of them invoke inflammation of keratinocytes in some way. We demonstrated in this study that activation of NF-κB is actively linked to cellular proliferation in keratinocytes which frequently originate from the external auditory canal skin. The molecular mechanism involves upregulation of cyclin D1 and downregulation p16Ink4a in keratinocytes. The former is a cell cycle progression protein and the latter is cell cycle progression inhibitor. Through this mechanism, Id1 opens a window for keratinocytes to actively grow and proliferate. Our in vitro studies demonstrate that PDTC, an inhibitor of NF-κB, blocks the Id1-induced proliferation of keratinocytes, indicating that Id1-induced cell proliferation is dependent upon NF-κB.
Activity of the cyclin D/retinoblastoma (Rb) pathway leads to proliferation of cells (25
). Cyclin D1, together with cyclin-dependent kinases 4/6 (cdk 4/6), overcomes the function of Rb protein that promotes cell cycle progression. It is generally accepted that normal Rb function must be removed, one way or another, for a cell to divide. Down-regulation of p16Ink4a
is one of the mechanisms for removing Rb function. Cyclin D1 is a well-established positive regulator of early cell cycle progression from G0/G1-to-S phase transition (26
) through phosphorylation of Rb and dissociation of E2F (a check point protein for S phase entry). The up-regulation of cyclin D1 and removal of p16Ink4a
inhibition in Rhek-1A cells by Id1 potentiates the growth and proliferation of keratinocytes and in part explains the behavior of keratinocytes in cholesteatoma— aggressive growth (27
) which destroys the ossicular chain and temporal bone and causes serious complications such as deafness and intracranial lesions. The Id1-induced cyclin D1 upregulation and p16Ink4a
downregualtion may represent a disease mechanism for cholesteatoma epithelial growth under chronic inflammatory conditions.
Activation of NF-κB not only increases the proliferation of keratinocytes but also upregulates the production of keratins. Keratin 10 is a product of mature keratinocytes (30
) and highly expressed in the external auditory canal skin. The abundant expression of keratin 10 suggests that keratinocytes in the middle ear cholesteatoma are originated from the external auditory canal epidermis possibly via a migration process under chronic inflammatory conditions (33
). Biologically, cell migration is coupled with cellular proliferation. Our data indicate that Id1 regulates NF-κB and NF-κB, in turn, upregulates the expression of keratin 10, which represents a pathological mechanism for accumulation of the onion peel-like substances in cholesteatoma. p65 strengthens this process whereas IκBαM attenuates this process.
It is noted that NF-κB at the suprabasal layer and above may not be related to cell growth and proliferation because cells at the suprabasal layer and above do not grow and proliferate but commit to keratin production. Referenced studies point out that NF-κB at the suprabasal layer and above may be related to survival and protection of cells from apoptosis (34
). Why Id1-induced NF-κB in the basal layer leads to cell proliferation while NF-κB alone beyond the basal layer results in cell survival and resistance to apoptosis is an interesting question. It is also not clear in this study how Id1 inhibits the expression of p16Ink4a
. It is warranted to address these questions in the future due to their importance in the pathogenesis of cholesteatoma and the physiology of the epidermis.