Id2 is dispensable for Myc-induced epidermal neoplasia

Abstract

We have previously described a transgenic mouse model of epidermal neoplasia wherein expression of a switchable form of c-Myc, MycER(TAM), is targeted to the postmitotic suprabasal keratinocytes of murine epidermis via the involucrin promoter. Sustained activation of c-MycER(TAM) results in a progressive neoplastic phenotype characterized by aberrant ectopic proliferation and delayed differentiation of suprabasal keratinocytes, culminating in papillomatosis. Transcription of the Id2 gene is regulated by Myc family proteins. Moreover, Id2 is implicated as a pivotal determinant of cell fate in multiple lineages and has a demonstrated role in mediating Myc-dependent cell proliferation in vitro through its interaction with retinoblastoma protein. Using Id2 nullizygous mice, we assessed in vivo the requirement for Id2 in mediating Myc-induced papilloma formation in skin. We show that absence of Id2 has no discernible impact on any measurable attribute of Myc function or on the timing or extent of eventual tumor formation. Thus, our data argue against any essential role for Id2 in mediating Myc action in vivo.

FIG. 1.

Id2 is expressed in hyperplastic Inv-MycER^TAM epidermis. Frozen sections of dorsal epidermis from wt (left panels) and Inv-MycER^TAM (right panels) mice treated for 2 weeks with 4-OHT were hybridized with ³⁵S-labeled antisense riboprobe specific for ODC-1, Id2, or Id4 mRNA. Dark-field microscopy revealed the presence of ODC-1 transcripts throughout all layers of the hyperplastic epidermis and a gradient of Id2 transcripts, highest in the inner (less differentiated) layers of the hyperplastic epidermis. Id4 transcripts by contrast were absent from normal and hyperplastic epidermis. Staining of hair cells was nonspecific.

FIG. 2.

Id2 is not required for the Myc-induced papillomatous phenotype. Hematoxylin and eosin (H&E) staining of Inv-MycER^TAM/Id2-wt and Inv-MycER^TAM/Id2-null epidermis after 7, 14, or 22 days of Myc activation. Inv-MycER^TAM-positive mice treated for 22 days with ethanol (top panels) and Inv-MycER^TAM-negative mice treated with 4-OHT (data not shown) comprised negative controls.

FIG. 3.

Myc-induced keratinocyte proliferation is unaffected by deletion of Id2. Inv-MycER^TAM/Id2-wt and Inv-MycER^TAM/Id2-null mice were treated with 4-OHT for 7, 14, or 22 days or with ethanol for 22 days. Expression of PCNA, indicative of cycling cells, is normally restricted to the basal layer of murine epidermis (top panels). DAB staining of keratinocyte nuclei reveals PNCA expression gradually extending through all layers of the suprabasal epidermis upon prolonged activation of MycER^TAM with 4-OHT.

FIG. 4.

Deletion of Id2 does not alter the delayed differentiation of suprabasal keratinocytes, induced by prolonged Myc activation. (A) Expression of keratin K10 in Inv-MycER^TAM/Id2-wt and Inv-MycER^TAM/Id2-null skin treated with 4-OHT for 7, 14, or 22 days. K10 expression normally initiates in the immediately suprabasal cell layer (top panels) but is delayed in onset and reduced in levels of expression after 14 days and especially after 22 days of continuous Myc activation. (B) Expression of keratin K14 in Inv-MycER^TAM/Id2-wt and Inv-MycER^TAM/Id2-null skin treated with 4-OHT for 7, 14, or 22 days. Keratin K14 identifies basal keratinocytes and keratinocyte stem cells. Because K14 is a relatively stable protein, DAB staining extends into the immediately suprabasal layer, but K14 is nonetheless lost in the outer layers of early hyperplastic epidermis (compare Myc “off” with Myc “on” at 7 days). Prolonged activation of Myc results in the retention of K14 protein throughout the late hyperplastic epidermis.

FIG. 4.

Deletion of Id2 does not alter the delayed differentiation of suprabasal keratinocytes, induced by prolonged Myc activation. (A) Expression of keratin K10 in Inv-MycER^TAM/Id2-wt and Inv-MycER^TAM/Id2-null skin treated with 4-OHT for 7, 14, or 22 days. K10 expression normally initiates in the immediately suprabasal cell layer (top panels) but is delayed in onset and reduced in levels of expression after 14 days and especially after 22 days of continuous Myc activation. (B) Expression of keratin K14 in Inv-MycER^TAM/Id2-wt and Inv-MycER^TAM/Id2-null skin treated with 4-OHT for 7, 14, or 22 days. Keratin K14 identifies basal keratinocytes and keratinocyte stem cells. Because K14 is a relatively stable protein, DAB staining extends into the immediately suprabasal layer, but K14 is nonetheless lost in the outer layers of early hyperplastic epidermis (compare Myc “off” with Myc “on” at 7 days). Prolonged activation of Myc results in the retention of K14 protein throughout the late hyperplastic epidermis.

FIG. 5.

Id2 is dispensable for Myc induction of dermal angiogenesis. The endothelial cell marker CD31 (FITC labeled) was used to identify total vasculature in the dermis of Inv-MycER^TAM/Id2-wt and Inv-MycER^TAM/Id2-null skin treated with 4-OHT for 14 or 22 days. Control tissue consisted of transgene-negative Id2-wt and Id2-null skin treated with 4-OHT for 22 days. Prolonged Myc activation in suprabasal epidermis results in an increase in the complexity and epidermal proximity of the underlying dermal vasculature irrespective of Id2 status.

FIG. 6.

Id1 and Id3 are abundantly expressed in Inv-MycER^TAM-induced epidermal hyperplasia. Inv-MycER^TAM/Id2-wt and Inv-MycER^TAM/Id2-null skin treated with 4-OHT for 14 days was stained for the presence of Id1 protein (top set of panels) or Id3 protein (bottom set of panels) by immunofluorescence. Alexa 488-conjugated secondary antibody (green), visible on the fluorescein isothiocyanate (FITC) channel, was used for detection, and sections were counterstained with propidium iodide (PI) (blue). Deletion of Id2 does not appreciably influence expression of Id1 or Id3 in the hyperplastic epidermis. Ab, antibody.