c-Myc promotes differentiation of human epidermal stem cells

Abstract

The epidermis contains two types of proliferative keratinocyte: stem cells, with unlimited self-renewal capacity, and transit amplifying cells, those daughters of stem cells that are destined to withdraw from the cell cycle and terminally differentiate after a few rounds of division. In a search for factors that regulate exit from the stem cell compartment, we constitutively expressed c-Myc in primary human keratinocytes by use of wild-type and steroid-activatable constructs. In contrast to its role in other cell types, activation of c-Myc in keratinocytes caused a progressive reduction in growth rate, without inducing apoptosis, and a marked stimulation of terminal differentiation. Keratinocytes can be enriched for stem or transit amplifying cells on the basis of beta1 integrin expression and by use of this method to fractionate cells prior to c-Myc activation, we found that c-Myc acted selectively on stem cells, driving them into the transit amplifying compartment. As a result, activation of c-Myc in epidermis reconstituted on a dermal equivalent led to premature execution of the differentiation program. The transcriptional regulatory domain of c-Myc was required for these effects because a deletion within that domain acted as a dominant-negative mutation. Our results reveal a novel biological role for c-Myc and provide new insights into the mechanism regulating epidermal stem cell fate.

Figure 1

Expression of c-Myc constructs and MycER/Max complex formation. (A) Western blot probed with anti-c-Myc antibody, 9E10. Cells were cultured in the absence of OHT. (Double-headed arrow) Position of 106ER (bottom) and major MycER (top) protein bands. (Single-headed arrow) Position of major wild-type c-Myc band. (B) Immunofluorescence staining of keratinocyte colonies maintained in the absence of OHT. Bar, 500 μm. (C) Keratinocytes were treated with OHT for 24 hr. Immunoprecipitation was performed with an anti-Max antibody (Mx) or preimmune serum (pre) and immunoblotted with anti-c-Myc (9E10). As a positive control, cell extracts were immunoblotted without prior immunoprecipitation (none). Arrows are as in A. Positions of molecular mass markers (200, 97.4, 69, 46 kD) are indicated by short horizontal lines in A and C.

Figure 2

Expression of members of the Myc network. Total RNA was subjected to Northern blotting with the probes shown. The top 18S panel is the control for c-Myc and Max; the bottom 18S panel is the control for Mad and Mxi-1. (Arrow) MycER or 106ER mRNA; (arrowhead) endogenous c-Myc mRNA.

Figure 3

Cell morphology. (A–G) Phase contrast micrographs of preconfluent (A–D) and confluent (E–G) keratinocytes. (A) KpBabe grown in the presence of OHT for 6 days; (B) KmycER, no OHT; (C) Kmyc, no OHT; (D) KmycER, 6 days OHT; (E) K106ER, 9 days OHT; (F) Kmyc, no OHT; (G) KmycER, 9 days OHT. Bar, 1 mm.

Figure 4

Growth curve. Cells were cultured in OHT throughout the experiment (▵ K106ER; (□) KpBabe; (○ KmycER). Data shown are means of triplicate dishes ±standard deviation.

Figure 5

Apoptosis. (A) Time-lapse video analysis of apoptosis. (Top) Cells were grown in complete medium (FAD, FCS, and HICE, i.e., including serum and growth factors) without OHT for 4 days, transferred to fresh medium, and then filmed (T.V.) for 2 days in the presence of OHT. (Bottom) Cells were grown in OHT throughout the experiment. After 1 day (asterisk), the medium was changed and replaced with either fresh complete medium (+ serum/GF) or FAD without FCS + HICE (− serum/GF). After another day, the medium was changed and filming began (T.V.). Filming continued for 4 days. The number of keratinocytes per field was scored at the beginning of each recording period (start) and the total number of apoptotic deaths (dead) per field is shown for each recording period. The final number of cells per field could not be scored accurately because of stratification but was estimated as ∼1000 for K106ER cells in complete medium and ∼500 for K106ER cells in FAD without supplements. The final number of KmycER cells per field was slightly lower than that of K106ER (see Fig. 4). (B) Determination of apoptosis by measurement of cytoplasmic histone-associated DNA in an ELISA. K106ER and KMycER were treated with OHT for 6 days. Cells were grown in complete medium (serum/GF) or starved in FAD alone for 30 h prior to extraction. Values are OD 405 nm. (Shaded boxes) +serum/GF; (solid boxes) −serum/GF.

Figure 6

Terminal differentiation. (A) Flow cytometric analysis of the proportion of involucrin-positive cells. No OHT, 5d (5 day) OHT and 9d (9 day) OHT panels: (Green lines) KpBabe; (blue lines) K106ER; (red lines) KmycER. Cells were harvested after culture in the presence or absence of OHT for the number of days shown. c-Myc panel: (Green line) KpBabe; (red line) Kmyc. Cells were grown in the absence of OHT. (y-axes) Cell number, (x-axes) fluorescence (arbitrary units, log scale). Positive staining for involucrin corresponds to fluorescence of >1.7 × 10¹ units. (B) Percent involucrin-positive cells measured in the absence of OHT or after addition of OHT for the number of days indicated. Cells from triplicate dishes were analyzed. Error bars show s.e.m.

Figure 7

Flow cytometry of KmycER (A,B) and K106ER (C,D) cultured for 10 days with addition of OHT, where indicated, for the final 3 days. (Solid lines) +OHT; (broken lines) −OHT. (A,C) labeled with anti-β₁ integrin antibody. (B,D) labeled with anti-α₃β₁ antibody. (A–D) (Dotted lines) Anti-CD8 (negative control). (y-axes) cell number. (x-axes) fluorescence in arbitrary units, log scale. Differentiated cells were gated out by forward and side scatter; fluorescence of basal (undifferentiated) cells is shown.

Figure 8

Clonogenicity of isolated stem and transit amplifying cells. K106ER and KMycER grown in the absence of OHT were harvested and labeled with an antibody to the β₁ integrin subunit; suprabasal cells were gated out and basal cells fractionated according to high or low integrin expression as indicated in the FACS profile. Cells were seeded at clonal density and cultured in the presence or absence of OHT. Duplicate 60-mm dishes are shown. Numbers are the percentage of abortive colonies (calculated from triplicate dishes). Insert below FACS profile shows an example of an actively growing clone (arrow) and an abortive clone (arrowhead) (×2.75).

Figure 9

In vitro-reconstituted epidermis. Sections from epidermis reconstituted by cells expressing 106ER (A,C,E,G) or MycER (B,D,F,H) in the presence of OHT. (A,B) hematoxylin/eosin staining; (C,D) staining with an anti-Ki67 antibody; (E,F) staining with anti-involucrin antibody; (G,H) staining with anti-filaggrin. Arrows in C and D indicate Ki-67-positive nuclei; arrows in F indicate involucrin-positive basal cells. Broken lines in G and H indicate position of basement membrane. Bar, 500 μm.