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NFAT proteins are calcium-regulated transcription factors that play a critical role during the timing and activation of many vertebrate tissues. A recent paper in Cell (Horsley et al., 2008) demonstrates a role of the calcineurin-NFAT-CDK4 pathway in maintaining hair follicle stem cell quiescence.
“Knowledge of the hair cycles and their control will undoubtedly give needed insight into many complicated growth processes of other body structures which are not at present well understood.”
—Earl O. Butcher (Butcher, 1934)
Despite their architectural and functional diversity, epithelial organs or appendages share common developmental strategies, including periodic self-renewal through the reactivation of multipotent progenitor cells. As Butcher foretold, the study of organ stem cell activation and regeneration has been a central issue in developmental biology and medicine for nearly a century. The hair is an ideal system to study the regulation of stem cell quiescence because stem cell identity and location (Morris et al., 2004; Tumbar et al., 2004) are well-characterized and the cells can be purified in large quantities. Hair follicles are easily visualized and possess an internal clock that allows them to cycle with fidelity about every 2 weeks in the mouse (about 2 years in humans) (Stenn and Paus, 2001).
Seventy-four years after the observations of Butcher, a paucity of information exists about the regulators of hair follicle quiescence. Intensive study of hair cycling has shown that proper growth and patterning require key morphogens such as Sonic hedgehog, Wnt, and bone morphogenetic protein (BMP) at the right times and places. One recent clue revealed that the BMP pathway plays a critical role in maintaining stem cell quiescence. Previous work by a number of labs demonstrated that loss of the BMP receptor or overexpression of BMP inhibitors can activate stem cells prematurely, although how BMP signaling regulates quiescence was not understood (Botchkarev and Sharov, 2004). A paper in a recent issue of Cell identifies the NFAT-calcium-CDK4 signaling pathway as a central regulator of stem cell quiescence and unifies several long-studied aspects of hair cycling (Horsley et al., 2008).
NFAT proteins are calcium-regulated transcription factors related to the Rel family that play a critical role during the timing and activation of many vertebrate tissues (Wu et al., 2007). The four major NFATc proteins are kept inactive in the cytoplasm by phosphorylation of their nuclear import signals by regulatory kinases such as Dyrk1, GSK3B, or PKA. Stimuli that raise intracellular calcium levels activate the trimeric calcium/cal-modulin-dependent phosphatase calcineurin to remove phosphatases from NFATc. Dephosphorylated NFATc is able to move into the nucleus where it dimerizes with various transcriptional partners (NFATn proteins) to induce growth factors, cytokines, and adhesion molecules. The relationship between calcineurin and NFAT signaling in the immune system has been exploited clinically to develop calcineurin inhibitors such as cyclosporine or FK506 that are potent immunosuppressive agents (Schreiber and Crabtree, 1992).
The role of NFATc as a signal integrator of widely varied pathways comes from at least three mechanisms. First, a wide variety of stimuli can raise intracellular calcium and activate calcineurin, including growth factor receptors, voltage-dependent ion channels, and gap junctions. Second, the DNA binding domain structure of NFATc requires interaction with other DNA binding domains for high-affinity binding. This requirement allows interactions with other signaling pathways at the level of DNA, thus connecting NFAT/calcium signaling to pathways such as AP1. Third, although NFAT activation occurs in response to growth factor signals, NFAT in turn induces a myriad of growth factors and receptors itself to amplify the initial signals.
Recent work culminates 2 decades of experimentation on the role of NFAT/calcineurin in hair follicle activation. Initial interest in calcium/calcineurin signaling in the epidermis came from the observation that increased calcium triggers epidermal differentiation but appears to inhibit hair follicle cycling. This calcium switch idea drew additional support from the initial observations that cyclosporine caused hair cycling apart from its effects on the immune system in mice and humans (Sawada et al., 1987). Further studies using conditional mutants implicated NFAT signaling in hair cycling by the demonstration that calcineurin B mutants showed cycling alopecia (Mammucari et al., 2005).
The present work shows that NFATc1 is the key NFAT regulating hair follicle stem cell quiescence. The epidermis lacking NFATc1 develops normally, but after initiating telogen, it prematurely enters the next anagen. The observation that stem cell and differentiation markers are not affected in the mutant mice suggests that NFATc1 acts specifically on hair cycling. The relationship of NFAT signaling to other hair follicle regulators is elucidated, as NFAT transcription appears to lie downstream of BMP signaling. Loss of BMP receptor results in loss of NFAT accumulation in the CD34+ stem cells. One difference between BMP and NFAT mutants is that, in BMPR1A mutants, the epithelium not only loses quiescence but also fails to differentiate. This suggests that BMP-mediated differentiation does not require NFATc1. Finally, the authors show that treatment with cyclosporine results in dramatic decreases in NFATc1 nuclear levels, consistent with its predicted mechanism of action. This elegant work shows that the NFAT-calcineurin pathway lies at the nexus of a variety of previously known hair follicle regulators (Figure 1).
Butcher predicted that knowledge of hair cycling would provide insight into the workings of other organs. Studies of NFAT, a well-characterized signaling pathway in the immune system, have revealed important details regarding hair cycling and have also stimulated many additional questions. Future studies will need to identify other environmental influences that effect pathway-mediated quiescence. In particular, because NFAT nuclear activity depends on intracellular calcium levels, a greater understanding is needed of calcium regulation in stem cells. Moreover, the role of other known hair cycle regulators, such as Sonic hedgehog or Wnt, in regulating stem cell cycling needs to be further elucidated. Wnt-induced stem cell activation occurs in the presence of nuclear NFAT, suggesting the existence of an NFAT-independent pathway for overcoming quiescence. Finally, BMP-dependent NFAT expression affects cycling, but not differentiation, arguing for the existence of an unidentified NFAT-independent differentiation pathway downstream of BMP. With the current pace of advances in understanding how hair follicle stem cells are regulated, studies of hair cycling will likely contribute greatly to studies of how other stem cells are regulated as well.