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. 2013 Jun 1;27(11):1217-22.
doi: 10.1101/gad.216556.113.

State-dependent Signaling by Cav1.2 Regulates Hair Follicle Stem Cell Function

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Free PMC article

State-dependent Signaling by Cav1.2 Regulates Hair Follicle Stem Cell Function

Gozde Yucel et al. Genes Dev. .
Free PMC article

Abstract

The signals regulating stem cell activation during tissue regeneration remain poorly understood. We investigated the baldness associated with mutations in the voltage-gated calcium channel (VGCC) Cav1.2 underlying Timothy syndrome (TS). While hair follicle stem cells express Cav1.2, they lack detectable voltage-dependent calcium currents. Cav1.2(TS) acts in a dominant-negative manner to markedly delay anagen, while L-type channel blockers act through Cav1.2 to induce anagen and overcome the TS phenotype. Cav1.2 regulates production of the bulge-derived BMP inhibitor follistatin-like1 (Fstl1), derepressing stem cell quiescence. Our findings show how channels act in nonexcitable tissues to regulate stem cells and may lead to novel therapeutics for tissue regeneration.

Keywords: VGCC; bulge; calcium channel; hair follicle stem cells.

Figures

Figure 1.
Figure 1.
The bulge lacks voltage-gated Ca+2 currents. (A) Immunofluorescent staining of bulge stem cells with Cav1.2 antibody (green) and CD34 antibody (red). (B) Topography of the channel Cav1.2, adapted from Splawski et al. (2004) with permission from Elsevier, © 2004. There are four segments, and each segment has six membrane-spanning domains. Here are shown the RT–PCR results of the main four segments. (P1) Brain cells from P1 mice; (B) bulge stem cells. Exons 4–7 (segment 1) are 400 base pairs (bp), exons 12–15 (segment 2) are 673 bp, exons 19–29 (segment 3) are 986 bp, and exons 30–37 (segment 4) are 514 bp. (C) FACS-sorted K15-EGFP bulge cells were plated on Matrigel-treated coverslips for whole-cell patch clamping experiments. (D) Raw traces of voltage clamp recording of Ba2+ currents from a single-cell bulge cell depolarized in −10-mV steps from a holding potential of −80 mV. Two-hundred-millisecond test depolarizations ranged from −100 to +50 mV; n = 8. (E) Raw traces of voltage clamp recording of K+ currents from a single-cell bulge cell depolarized in −10-mV steps from a holding potential of −80 mV. Two-hundred-millisecond test depolarizations ranged from −100 to +40 mV; n = 8. (F) Ratiometric Ca2+ imaging was carried out using Fura-2. Cells were depolarized with 65 mM KCl to elicit voltage-gated Ca2+ entry and treated with thapsigargin (Tg) in the absence of extracellular Ca2+ to empty ER-Ca2+ stores, followed by readdition of 2 mM Ca2+ to visualize store-operated Ca2+ entry. Averaged Fura-2 responses of bulge cells (blue) and control cells (red); n = 39 for control cells; n = 11 for bulge cells. (G) Representative single-cell curves for both cell types showing cell-to-cell variability. (H) Fura-2 signals at 5 mM K (Low K), after depolarization at 65 mM K (High K), and after store depletion followed by Ca2+- addition (Post-Tg).
Figure 2.
Figure 2.
Persistent telogen in Cav1.2TS mice and in pore mutants. (A) RU486 treatment strategy and C57BL6 animals' hair cycling times. (A) Anagen; (T) telogen; (C) catagen. (B) Cav1.2TS mice are still in telogen (right), while control mice are already in anagen (left). Pink skin represents telogen, and dark skin indicates anagen. Hematoxylin and eosin staining of control wild-type (WT) skin (C) and Cav1.2TS skin (D). Bar, 100 μm. (E) Immunofluorescence staining of Nfatc1 of control and Cav1.2TS bulge cells. Bar, 20 μm. (F) BrdU staining of control and Cav1.2TS bulge cells. Bar, 20 μm. (G) Ki67 staining of control and Cav1.2TS skin. Bar 100 μm. (H) Cav1.2Δ14–15 mutant mice (right) have extended telogen compared with control mice (left). Hematoxylin and eosin staining of control skin (I) and Cav1.2Δ14–15 mutant skin (J). Bar, 100 μm.
Figure 3.
Figure 3.
L-type channel blockers act oppositely to pore mutants. (A) Verapamil treatment strategy. (B) Verapamil-treated mice go into anagen earlier (right) than control littermates (left). (VER) verapamil; (VEH) vehicle. Hematoxylin and eosin staining of control (C) and treated (D) skin. Bar, 100 μm. (E) Ki67 staining of control and verapamil-treated skin. Bar, 100 μm. (F) Ki67 staining of control and verapamil-treated Cav1.2Δ14–15 mutant skin. Bar, 100 μm. (G) Verapamil-treated Cav1.2Δ14–15 mutant mice (right) go into anagen later than their control littermates (left). Hematoxylin and eosin staining of control skin (H) and verapamil-treated Cav1.2Δ14–15 mutant skin (I). Bar, 100 μm.
Figure 4.
Figure 4.
L-type channel blockers regulate anagen by inducing Fstl1. (A) A heat map of microarray results of Cav1.2Δ14–15 mutant and control bulge stem cells. The first two rows are genes from mutant bulge cells (M), and the last two rows are genes from wild-type bulge cells (W). (B) Other BMP inhibitors go down or do not change in verapamil-treated bulge stem cells analyzed by quantitative PCR (qPCR). qPCR results of Fstl1 in control and verapamil-treated bulge cells (C) and control and Cav1.2Δ14–15 mutant bulge cells (D). (E) Immunofluorescence staining of Fstl1 in control, verapamil-treated, and verapamil-treated Cav1.2Δ14–15 mutant bulge. Bar, 20 μm. (F) Injection of Fstl1 protein-soaked beads caused early entry to anagen. The mouse at the left received a full dose (4 μg) of Fstl1 protein, and the mouse in the middle received a half dose (2 μg) of Fstl1. The mouse at the right received 4 μg of BSA. Red arrows point to the injection site. (GI) Hematoxylin and eosin staining of skin of the mice from F. Bar, 100 μm. (J) Model of Cav1.2 in murine hair cycling. During telogen, Cav1.2 is in the closed state, and BMPs are expressed. This state is represented by the Cav1.2Δ14–15 loss-of-function mutant. The open state is represented by the Cav1.2TS mutant, and this state is the same as the closed state in hair follicle stem cells. Verapamil binding will put Cav1.2 into the inactive state, which in turn will activate Fstl1, which blocks BMPs and lets anagen begin.

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