Myo/Nog Cells in Normal, Wounded and Tumor Bearing Skin

Abstract

Murine and human skin were examined for the presence of Myo/Nog cells that were originally discovered in the chick embryo by their expression of MyoD mRNA, noggin and the G8 antigen. Myo/Nog cells are the primary source of noggin in telogen hair follicles. They are scarce within the interfollicular dermis and absent in the epidermis. Within 24 hours following epidermal abrasion, Myo/Nog cells increase in number in the follicles and appear in the wound. Myo/Nog cells are also recruited to the stroma of tumors formed from v-Ras transformed keratinocytes (Ker/Ras). Human squamous cell carcinomas and malignant melanomas contain significantly more Myo/Nog cells than basal cell carcinomas. Myo/Nog cells are distinct from macrophages, granulocytes and cells expressing alpha smooth muscle actin in the tumor stroma. Myo/Nog cells may be modulators of skin homeostasis and wound healing, and potential diagnostic and therapeutic targets in skin cancer.

BACKGROUND

Bone morphogenetic proteins (BMPs) play a key role in controlling epidermal homeostasis, hair follicle growth, melanogenesis, wound healing and tumorigenesis [1–12]. The activity of BMPs is regulated, in part, by secreted BMP antagonists, including noggin [13]. Noggin is required for hair follicle induction and morphogenesis in the embryo and the promotion of new hair growth in the adult [14, 15]. However, overexpression of noggin in the skin results in spontaneous development of trichofolliculoma-like tumors [16]. Thus, modulation of BMP signaling must be tightly regulated to maintain normal skin architecture and function.

We discovered a cell that modulates BMP signaling and participates in wound healing in the chick embryo [17–20]. “Myo/Nog” cells, identified by their expression of mRNA for the skeletal muscle specific transcription factor MyoD, noggin and the G8 cell surface antigen, are critical regulators of morphogenesis and differentiation [17–19, 21, 22]. Chick embryo Myo/Nog cells also respond to cells undergoing apoptosis and tissue wounding by rapidly expanding and migrating to the site of injury [17, 20].

QUESTIONS ADDRESSED

We investigated whether Myo/Nog cells are present in the skin of adult mice and humans and if they respond to epidermal abrasion and tumorigenesis.

EXPERIMENTAL DESIGN

Normal and abraded skin of FVB mice were embedded in paraffin [23]. V-Ras transformed mouse keratinocytes (Ker/Ras) were injected subcutaneously and tumors excised 11 weeks later [24, 25]. Sections of human skin were examined in tissue arrays and slides of breast tissue and squamous cell carcinoma. Myo/Nog and other cell types were identified by immunofluorescence localization and in situ hybridization [18, 21].

RESULTS

Myo/Nog cells were identified in the secondary hair germ of telogen phase follicles of murine skin by their co-expression of the G8, MyoD mRNA and noggin mRNA and protein. In each section, 25–50% of the follicles contained 2–4 Myo/Nog cells (Figs. 1C, S1C). Anagen phase follicles had more Myo/Nog cells than telogen follicles (Fig. S1H). They were rarely found in the interfollicular dermis and none were observed within the epidermis (Figs. 1E, S1E). Within 24 hours of epidermal abrasion, the number of G8+/MyoD+/noggin+ cells had increased within and surrounding the follicles (Figs. 1D, S1D). Clusters of Myo/Nog cells were present in the wounded surface of the dermis (Figs. 1F, S1F).

Figure 1. Myo/Nog cells in normal, abraded and tumor bearing murine skin.

Skin sections were stained with H&E (A, B and G) or double labeled for G8 and noggin mRNA (Nog) or noggin protein (inset in C). Insets in A and B illustrate the areas shown at high magnification in the fluorescence photomicrographs. The colors of the fluorescent tags are indicated in each photograph. Nuclei were stained with Hoechst dye. Overlap of green and red appears yellow in merged images. The hair shaft and keratin within the tumor are autofluorescent (*). In unabraded skin (A), Myo/Nog cells were located in the secondary hair germ (f) (C). Inset in B shows Myo/Nog cells within a follicle sectioned transversely. Myo/Nog cells were not observed in the interfollicular dermis (d) or epidermis (e) (C and E). Twenty four hours after abrasion (B), Myo/Nog cells were increased in the follicles and surrounding dermis (D) and were present at the wounded surface (F). Ker/Ras tumors (G) contained Myo/Nog cells in the stroma (H). Some Myo/Nog cells surrounded TUNEL+ cells (I). Myo/Nog cells were increased in the follicles overlying the tumor (J). Bar = 56 µm in A, B and G, and 9 µm in C-F and H-J.

Myo/Nog cells were also found in the stroma of Ker/Ras tumors (Figs. 1H, S2B). Some were adjacent to TUNEL+ cells (Fig. 1I). Only a few Myo/Nog cells had invaded the tumor epithelial compartment. Markers for monocytes/macrophages (F480), myofibroblasts (alpha smooth muscle actin (α-SMA)) and squamous cell carcinoma and mesenchymal stem cells (CD44) were not detected in Myo/Nog cells (Fig. S2). Follicles overlying tumors contained increased numbers of Myo/Nog cells (Figs. 1J, S2C). Less than 1% of the stained cells in double labeled sections of normal, abraded and tumor bearing skin contained G8, MyoD or noggin mRNA alone.

In human skin tumors, all G8+ cells expressed MyoD mRNA and 73% were co-stained for noggin (Fig. 2). Whereas Myo/Nog cells were rarely found in the normal dermis, they were present in clusters underlying basal cell carcinoma cells (Fig. 2B). In squamous cell carcinomas, Myo/Nog cells were concentrated within the stromal compartment; however, some were present in the tumor itself (Fig. 2D,F). A greater degree of Myo/Nog cell infiltration among tumor cells was observed in malignant melanomas than the skin carcinomas (Fig. 2G,H). Some Myo/Nog cells appeared to have melanin in the cytoplasm (Fig. 2H).

Figure 2. Myo/Nog cells in normal human skin and tumors.

Sections of basal cell carcinoma (A, B), squamous cell carcinoma (C-F) and malignant melanoma (G and H) were stained with H&E or double labeled for G8 and either MyoD mRNA or noggin protein (Nog). Arrows in H&E stained sections indicate the approximate areas shown in high magnification fluorescent images. The colors of the fluorescent tags are indicated in each photograph. Nuclei were stained with Hoechst dye. Overlap of green and red appears yellow in merged images. Merges of DIC and fluorescent images are shown in G and H. In squamous cell carcinoma, Myo/Nog cells were present in the stroma (D) and among the cancer cells (F). Myo/Nog cells surrounded pigmented melanoma cells (arrows in G). Some Myo/Nog cells were pigmented (arrows in I). Bar = 27 µm in A, C and E, and 9 µm in B, D and F-H.

Significantly more Myo/Nog cells were found in squamous cell carcinomas than melanomas (p = 0.0185) and in both squamous cell carcinomas and melanomas than basal cell carcinomas or normal dermal tissue (p < 0.0001) (Supplement Table 1). Variation in Myo/Nog cell number was observed in sections from the same subject, indicating that they are not uniformly distributed in tumors. The number of G8+ cells correlated with the extent of tumor invasion in squamous cell carcinomas (T value) (p < 0.0001) (Supp. Table 1). α-SMA, cytokeratins and CD44 were not detected in Myo/Nog cells in human tumors (Fig. S3).

CONCLUSIONS

In this study we revealed the presence of Myo/Nog cells in skin of adult mice and humans. They appear to be the primary source of noggin in the hair follicle, wounded dermis and tumor stroma, as determined by co-localization of noggin mRNA with G8. Although G8 and α-SMA were detected in separate populations in tumors, the G8 antigen is downregulated as α-SMA accumulates in embryonic cells [20]. Therefore, Myo/Nog cells may be a source of at least some α-SMA+ myofibroblasts that are also recruited to wounds and tumors [26, 27]. Myo/Nog cells do appear to be distinct from ranulocytes and macrophages based on their unsegmented nuclei and absence of F4/80 staining.

Myo/Nog cells were more abundant in malignant melanomas and squamous cell carcinomas than basal cell carcinomas, suggesting that the extent to which Myo/Nog cells populate tumors may correlate with metastatic potential. In melanomas, a few Myo/Nog cells contained melanin. Myo/Nog cells may have been induced to synthesize melanin, engulfed melanosomes, phagocytosed apoptotic melanoma cells or fused with melanoma cells in a phenomenon akin to the cannibalism of lymphocytes by melanoma cells [28].

Blocking BMP signaling promotes the development of skin tumors in mice and the growth of human melanoma cells [6, 11, 12, 16, 29]. However, BMPs stimulate the proliferation of other types of cancer cells [30]. Therefore, the effect of noggin is expected to be context dependent and ultimately reflect how tumor and Myo/Nog cells integrate their inputs from multiple signaling pathways. Dissection of the full repertoire of Myo/Nog cell functions will be important for determining whether they are useful targets for diagnosing, prognosing and treating cancer.