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First published online 8 April 2008
doi: 10.1242/jcs.018689

Journal of Cell Science 121, 1435-1443 (2008)
Published by The Company of Biologists 2008
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Control of thrombin signaling through PI3K is a mechanism underlying plasticity between hair follicle dermal sheath and papilla cells

Anne-Catherine Feutz1,*, Yann Barrandon2 and Denis Monard1,{ddagger}

1 Friedrich Miescher Institute for Biomedical Research, CH-4058, Basel, Switzerland
2 Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne and Lausanne University Hospital, Station 15, CH-1015 Lausanne, Switzerland


Figure 1
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  		Fig. 1. PN-1 colocalizes with thrombin and controls its activity in vibrissal follicles. (A) X-Gal (blue) staining of cryostat sections from embryonic hair (E18.5) and whisker (E16.5) follicles of PN-1 knockin (KI) mice during early follicle morphogenesis. (B) Immunohistochemistry (brown staining) of follicles during anagen (P23) and catagen (P25) with an antibody against PN-1 (note that black areas are melanin, not staining). (C) X-gal staining of adjacent longitudinal cryostat sections of dissected postnatal follicles from a PN-1 KI mouse at early anagen (P27). (D) Thrombin immunostaining of a longitudinal cryostat section of a full-length early anagen follicle from a wild-type mouse showing the similarity to PN-1 localization (cf. C) in DS and a subset of ORS cells. (E) Comparison of thrombin proteolytic activity with or without addition of recombinant PN-1 (rPN-1; 1 µg/ml) to anagen whisker follicle homogenates from wild-type and PN-1–/– mice. ***Significant difference (P<0.001). (F) Higher magnification of thrombin immunostained follicular bulbs from wild-type and PN-1–/– mice showing thrombin accumulation in the DP in the absence of PN-1. pDP, presumptive dermal papilla; pDS, presumptive dermal sheath; DP, dermal papilla; DS, dermal sheath; ORS, outer root sheath; C, capsula.

 

Figure 2
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  		Fig. 2. Cultured DP cells exhibit characteristics of both DP and DS cells. Immunostaining of cultured wild-type DP cells from microdissected dermal papilla. (A) PN-1, (B) LDL receptor-related protein-1 (LRP-1), (C) thrombin receptor PAR-1, (D) NCAM, (E) alkaline phosphatase (AP), (F) smooth muscle actin (SMA) and BrdU (10 µM, 2 hours).

 

Figure 3
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  		Fig. 3. PN-1-mediated inhibition of thrombin activity affects proliferation of cultured hair follicle dermal cells. (A) Growth of wild-type and PN-1–/– DP cell in medium containing 10% FCS. Cells were plated at 200 cells/mm2 and average cell density from three dishes was calculated at different time points after plating. (B) Effect of hirudin-mediated inhibition of thrombin in serum-supplemented culture medium on cell proliferation. Hirudin (100 µM) was added 24 hours before a pulse of BrdU (10 µM, 2 hours). ***Significant difference from wild-type cells (P<0.001). (C) Effect of PN-1 on volume increase. Cells transferred to medium containing 1% FCS were blocked in S phase by aphidicolin (1 µg/ml) for 24 hours and then cultured for an additional 24 hours with or without recombinant PN-1 (rPN-1; 15 nM) in the absence or presence of RAP (1 µg/ml). Change in mean volume was calculated for non-dividing cells in three dishes for each treatment. **Significant difference between PN-1–/– and wild-type cells (P<0.01); *significant difference between PN-1–/– cells with or without rPN-1 (P<0.05).

 

Figure 4
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  		Fig. 4. PN-1 protects DP cells from thrombin-stimulated expression of a myofibroblastic phenotype. (A) Level of SMA protein in cultured wild-type and PN-1–/– DP cell lysates (upper panel) and in extracts from the hairy skin of wild-type and PN-1–/– mice (unplucked; unP) and excised 3 days after stimulation of SMA expression by hair plucking (P) (lower panel). (B) Collagen gel contraction by embedded wild-type and PN-1–/– DP cells after 48 hours in medium containing 10% FCS. ***Significant difference from wild-type cells (P<0.001). (C) Effect of thrombin inhibition by hirudin (100 µM) on gel contraction by DP cells. (D) Effect of PAR-1-activating peptide (TRAP; 600 µM) or retro-sequence control peptide (600 µM) on the maintenance of contractile properties of wild-type DP cells transferred to a medium containing 1% serum. **Significant difference from 1% FCS (P<0.01).

 

Figure 5
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  		Fig. 5. Thrombin stimulates both growth and contractibility through activation of the PI3K/Akt pathway. (A) Percentages of wild-type and PN-1–/– (KO) cells incorporating BrdU (10 µM, 2 hours). The PI3K inhibitor LY294002 (LY; 50 µM) and the mTOR inhibitor RAD001 (RAD; 20 nM) were added in 10% FCS medium 24 hours before BrdU incorporation. ***Significant difference from wild-type cells (P<0.001). (B) Volume increase of cells switched to 1% FCS medium and arrested with aphidicolin (1 µg/ml, as in Fig. 3) determined after 24 hours with or without LY294002 or RAD001. *Significant difference from wild-type cells (P<0.05). (C) Effects of LY294002 or GF103203X (5 µM) on thrombin-stimulated (1 U/ml) collagen gel contraction by wild-type and PN-1–/– (KO) DP cells pretreated with hirudin (100 µM, 6 hours) and cultured in the presence of hirudin and aphidicolin. *Significant difference from wild-type cells (P<0.05). (D) Relative levels of phospho-Akt and total Akt proteins in extracts from wild-type and PN-1–/– DP cells assessed by immunoblotting with the indicated antibodies. Cells were either untreated (/) or cultured in the presence of LY294002 or RAD001 as in A. (E) Effect of thrombin on Akt phosphorylation examined by adding thrombin (1 U/ml, 30 minutes) to wild-type DP cells grown in medium containing 1% FCS. The dependence of the thrombin effect on PI3K pathway was assayed by adding LY294002 or RAD001 1 hour prior to thrombin. (F) Involvement of PAR-1 in Akt activation. Wild-type DP cells were treated with thrombin (as in E), TRAP or control peptides (as in Fig. 4). (G) Comparison of the efficiency of thrombin stimulation of Akt phosphorylation in wild-type versus PN-1–/– DP cells tested as in D. (H) Phosphorylation status of proteins of the PI3K pathway in lysates from dissected anagen whisker follicles of 20-day-old wild-type and PN-1–/– littermates.

 

Figure 6
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  		Fig. 6. Absence of PN-1 exacerbates the detrimental effects of thrombin on the ability of the DP cells to support keratinocytes. (A,B) Effect of DP cells on the growth and survival of keratinocytes. (A) Clusters formed after 8 days by YF29 keratinocytes plated at 15,000 cells/cm2 on inactivated wild-type or PN-1–/– DP feeder cells and visualized by Rhodamine B staining. (B) Capacity of DP cells to sustain cluster formation, quantified by counting clusters of different size ranges. ***Significant difference from wild-type cells (P<0.001). (C) Immunoblot evaluation of Akt phosphorylation in keratinocyte to monitor the signaling triggered by DP cells secreted molecules. YF29 keratinocytes were maintained in minimal medium (control) or transferred for 30 min to minimal medium conditioned for 24 hours by untreated DP cells (WT, KO for PN-1–/–) or by cells pretreated with thrombin at 1 U/ml (WT+, KO+).

 

Figure 7
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  		Fig. 7. In vivo wild-type dermal papilla expresses the DS marker SMA when switched from anagen to catagen. (A) SMA expression in lysates of dissected whisker follicles in anagen A and catagen C. (B) Immunostaining of cryostat sections of wild-type back skin. SMA expression in the DS of hair follicles in anagen (P12) and in the DS and DP in catagen (P15). (C) Co-expression of SMA and AP in DP cells of wild-type back skin during catagen (P15). Left image shows AP staining alone for comparison. SMA, alpha smooth muscle cells actin; DS, dermal sheath; AP, alkaline phosphatase; DP, dermal papilla; TS, trailer sheath.

 

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