Differential expression of cyclin D1 in the human hair follicle.
Journal: 2003/September - American Journal of Pathology
ISSN: 0002-9440
Abstract:
The proliferation of keratinocytes in the hair follicle varies from slowly cycling, intermittently proliferating stem cells in the bulge to rapidly proliferating, transient cells in the bulb. To better understand the biological differences between these two compartments, we sought to identify differentially expressed genes using cDNA macroarray analysis. Cyclin D1 was one of 13 genes increased in the bulge compared to the bulb, and its differential expression was corroborated by quantitative real-time polymerase chain reaction (PCR) on the original samples. Using immunohistochemical staining, laser-capture microdissection (LCM) and quantitative real-time PCR, we localized cyclin D1 to the suprabasal cells of the telogen bulge and anagen outer root sheath (ORS). Surprisingly, cyclin D1, D2, and D3 were not detectable by immunohistochemistry in the rapidly proliferating hair-producing cells of the anagen bulb (matrix cells), while these cells were strongly positive for Ki-67 and retinoblastoma protein. In contrast, pilomatricoma, a tumor thought to be derived from matrix cells, was positive for cyclin D1, D2, and D3. Our results suggest that cyclin D1 may mediate the proliferation of stem cells in the bulge to more differentiated transient amplifying cells in the suprabasal ORS. In contrast, non-cyclin D1-proteins appear to control cell division of the highly proliferative bulb matrix cells. This non-cyclin D1-mediated proliferation may provide a protective mechanism against tumorigenesis, which is overridden in pilomatricomas. Our data also demonstrate that the combination of DNA macroarray, LCM and quantitative real-time PCR is a powerful approach for the study of gene expression in defined cell populations with limited starting material.
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Am J Pathol 163(3): 969-978

Differential Expression of Cyclin D1 in the Human Hair Follicle

From the Departments of Pathology and Dermatology, Hospital of University of Pennsylvania, Philadelphia, Pennsylvania; and the Departments of Pathology and Dermatology, Harvard Medical School, Boston, Massachusetts
Accepted 2003 May 27.

Abstract

The proliferation of keratinocytes in the hair follicle varies from slowly cycling, intermittently proliferating stem cells in the bulge to rapidly proliferating, transient cells in the bulb. To better understand the biological differences between these two compartments, we sought to identify differentially expressed genes using cDNA macroarray analysis. Cyclin D1 was one of 13 genes increased in the bulge compared to the bulb, and its differential expression was corroborated by quantitative real-time polymerase chain reaction (PCR) on the original samples. Using immunohistochemical staining, laser-capture microdissection (LCM) and quantitative real-time PCR, we localized cyclin D1 to the suprabasal cells of the telogen bulge and anagen outer root sheath (ORS). Surprisingly, cyclin D1, D2, and D3 were not detectable by immunohistochemistry in the rapidly proliferating hair-producing cells of the anagen bulb (matrix cells), while these cells were strongly positive for Ki-67 and retinoblastoma protein. In contrast, pilomatricoma, a tumor thought to be derived from matrix cells, was positive for cyclin D1, D2, and D3. Our results suggest that cyclin D1 may mediate the proliferation of stem cells in the bulge to more differentiated transient amplifying cells in the suprabasal ORS. In contrast, non-cyclin D1-proteins appear to control cell division of the highly proliferative bulb matrix cells. This non-cyclin D1-mediated proliferation may provide a protective mechanism against tumorigenesis, which is overridden in pilomatricomas. Our data also demonstrate that the combination of DNA macroarray, LCM and quantitative real-time PCR is a powerful approach for the study of gene expression in defined cell populations with limited starting material.

Abstract

Hair grows in a regulated cyclical process consisting of three distinct phases: anagen, catagen, and telogen. In human scalp follicles, anagen, the growing phase, lasts several years; catagen, the involution phase, lasts approximately 1 to 3 weeks, and telogen, the resting phase, lasts for approximately 3 months. The cellular mechanisms involved in the maintenance of the phases and the transitions between them are poorly understood. The lack of progress in this area reflects the complicated structure and physiology of the hair follicle. In addition, human hair follicles grow in an asynchronous fashion; the majority (∼90%) of hair follicles are in the anagen phase and less than 10% of follicles are in telogen, thus making it difficult to study the hair follicle cycle in humans. However, understanding what controls the proliferation of the follicle could have wide-ranging implications for carcinogenesis and hair disorders.

One important aspect of the transition from the telogen phase to the anagen phase is the mechanism which promotes stem cell proliferation. The stem cells of the bulge region are normally quiescent throughout catagen, telogen and most of the anagen phase but proliferate briefly at anagen onset. In contrast, the anagen matrix cells, which are considered “transient amplifying” (TA) cells, proliferate constantly during anagen. In fact, these cells display one of the highest proliferative rates of any mammalian tissue, with a growth fraction of almost 100%, even outranking most malignant tumors. As in many other highly proliferative tissues, such as bone marrow and gastrointestinal epithelium, this may represent a risk for mutagenesis. But malignant tumors derived from matrix keratinocytes are exceedingly rare; although benign tumors, such as pilomatricomas, occasionally arise from hair matrix cells.

In mammalian cells, proliferation is under the control of factors that regulate the transitions between different cell-cycle stages at two main checkpoints. The better-characterized checkpoint is at the G1-S transition, which initiates DNA replication in S phase. The other checkpoint is at the G2-M transition, which controls mitosis and cell division. The cyclins are a family of key cell-cycle regulators that function by association with and activation of cyclin-dependent kinases (CDKs) at specific points in the cell cycle to phosphorylate various proteins that are important during cell cycle progression. A key substrate for G1 cyclins/CDK complexes is the retinoblastoma tumor suppressor protein (RB). The phosphorylation of RB then releases E2F, which is important in the initiation of DNA replication and in the G1-S phase transition. The levels of cyclins are regulated at the level of transcription as well as by targeted degradation via the ubiquitin pathway.

β-catenin plays an intricate role in Wnt signaling. β-catenin regulates gene expression by direct interaction with Lef-1, providing a molecular mechanism for the transmission of signals, from cell-adhesion components or Wnt protein to the nucleus. Lef1/β-catenin has been identified as a key regulator in hair follicle differentiation and development. The cyclin D1 gene is a direct target for transactivation by the β-catenin/LEF-1 pathway through a LEF-1 binding site in the cyclin D1 promoter and therefore a direct downstream molecule in the β-catenin pathway.

To better characterize the transition from quiescent stem cells to proliferative daughter TA cells, we used DNA macroarrays to compare gene expression in the bulge cells to the bulb matrix cells. We identified cyclin D1 as one of the genes up-regulated in the telogen bulge compared to the anagen bulb. Localization of cyclin D1 expression in the hair follicle using laser-capture microdissection (LCM), real-time polymerase chain reaction (PCR) and immunohistochemical staining suggests that it may play a role in conversion of stem cells to transient amplifying cells, but not in the maintenance of the continual hair follicle proliferation required for hair production.

Housekeeping genes.

The approximate ratios of the signals after normalization to housekeeping genes are listed in the third column.

Acknowledgments

We thank Dr. James Eberwine for the use of his LCM apparatus, Dorothy Campbell and the histology staff of the Departments of Pathology and Dermatology at the University of Pennsylvania for technical assistance. The Cooperative Human Tissue Network, which is funded by the National Cancer Institute, provided tissue specimens.

Acknowledgments

Footnotes

Address reprint requests to George Cotsarelis, M.D., Department of Dermatology, Hospital of University of Pennsylvania, M8 Stellar Chance Building, 422 Curie Boulevard, Philadelphia, PA 19104. E-mail: .ude.nnepu.dem.liam@lerastoc

Supported by National Institutes of Health grants R29-AR-44038 and R01-AR46837, and grants from the National Alopecia Areata Foundation.

The abstract of this paper received the Stowell-Orbison Award from the United States and Canadian Academy of Pathology.

Footnotes

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