A Cell Membrane-Level Approach to Cicatricial Alopecia Management: Is Caveolin-1 a Viable Therapeutic Target in Frontal Fibrosing Alopecia?

Abstract

Irreversible destruction of the hair follicle (HF) in primary cicatricial alopecia and its most common variant, frontal fibrosing alopecia (FFA), results from apoptosis and pathological epithelial-mesenchymal transition (EMT) of epithelial HF stem cells (eHFSCs), in conjunction with the collapse of bulge immune privilege (IP) and interferon-gamma-mediated chronic inflammation. The scaffolding protein caveolin-1 (Cav1) is a key component of specialized cell membrane microdomains (caveolae) that regulates multiple signaling events, and even though Cav1 is most prominently expressed in the bulge area of human scalp HFs, it has not been investigated in any cicatricial alopecia context. Interestingly, in mice, Cav1 is involved in the regulation of (1) key HF IP guardians (TGF-β and α-MSH signaling), (2) IP collapse inducers/markers (IFNγ, substance P and MICA), and (3) EMT. Therefore, we hypothesize that Cav1 may be an unrecognized, important player in the pathobiology of cicatricial alopecias, and particularly, in FFA, which is currently considered as the most common type of primary lymphocytic scarring alopecia in the world. We envision that localized therapeutic inhibition of Cav1 in management of FFA (by cholesterol depleting agents, i.e., cyclodextrins/statins), could inhibit and potentially reverse bulge IP collapse and pathological EMT. Moreover, manipulation of HF Cav1 expression/localization would not only be relevant for management of cicatricial alopecia, but FFA could also serve as a model disease for elucidating the role of Cav1 in other stem cell- and/or IP collapse-related pathologies.

Keywords: caveolin-1; cicatricial alopecia; cyclodextrin; frontal fibrosing alopecia; hair follicle; immune privilege; statin.

Figure 1

Proposed mechanism for the role of Caveolin-1 in development of FFA. (a) Inflammation-induced downregulation of E-cadherin along with excessive IFNγ and EGF signaling promote pathological EMT in bulge epithelial cells of human scalp HFs. (b) Upregulation of caveolin-1 (Cav1) expression allows for monomeric Cav1 to oligomerize at the cell membrane and induce formation of caveolae. Cav1 antagonizes TGF-β signaling by sequestering TGF-β receptors and preventing phosphorylation and signaling through Smad2, inhibiting its association with Smad4, as well as subsequent Smad2 nuclearization and TGFβ-mediated transcriptional activation. Conversely, upregulation of Cav1 leads to increased Substance P levels and promotes localization of its receptor NK-1 to caveolae where NK-1 interacts with downstream G-protein, resulting in sustained Substance P signaling through the NK-1 receptor. Lastly, upregulation of Cav1 has been associated with upregulation of MICA and Vimentin, as well as a downregulation of E-cadherin. Therefore, upregulation of Cav1 orchestrates development of environment permissive to eHFSC IP collapse by (1) inhibiting guardians of IP (TGF-β signaling), (2) promoting suppressors of IP (Substance P and MICA), and (3) promoting EMT (upregulating vimentin and downregulating E-cadherin).

Figure 2

Caveolin-1 colocalizes with markers of eHFSCs in the outer root sheath cells in human hair follicles. (a) Cav1 immunostaining of normal human scalp hair follicles exhibits colocalization with K15 and CD34, common markers of eHFSC in ORS cells. ORS-Outer Root Sheath. Treatment of normal human scalp hair follicles with Sandalore results in downregulation of Cav1 expression in the bulge at both protein (b,c) and mRNA levels (d). Data are expressed as mean ± SEM, n = 13–16 HFs from 2 different donors, Student’s t-test, *** p < 0.001, GraphPad Prism 6.

Figure 3

Elevated levels of Cav1 in FFA. (a) FFA scalp at the level of the bulge exhibits upregulation of Cav1 expression in comparison to normal scalp from the healthy human donors. HF: Hair follicle; SG: Sebaceous gland. (b) Quantification of Cav1 expression from basal layer of outer root sheath cells with error bars corresponding to SEM from n = 3 different donors. (c) Levels of other structural components of caveolae (Cav2 and Cavin1/PTRF) remain unchanged in comparison to healthy normal scalp, thus indicating a Cav1-specific effect.

Figure 4

Differential regulation of IP Guardian/Collapse and EMT/MET-related genes in Cav1 knockout mouse skin. (a) Full thickness skin biopsy punches from location matched 8-week-old female C57BL6 (i.e., Cav1 WT type) and global Cav1 knockout (Cav1^KO) mice were utilized to assess expression levels of IP Guardian (CD200, IL-10), IP Collapse (Substance P, β2MG, MHC Class I, CXCL11), and EMT/MET-related genes (E-cadherin, N-cadherin, TWIST1) by qRT-PCR. Cav1^KO mouse skin exhibits upregulation of IP guardian genes and a marker of epithelial cells (E-cadherin), as well as a downregulation of IP collapse and EMT related genes. Error bars correspond to standard deviation from n = 3 mice.

Figure 5

Lovastatin treatment of Outer Root Sheath (ORS) keratinocytes results in downregulation of numerous structural components of caveolae. Primary Outer Root Sheath (ORS) keratinocytes isolated from normal human scalp were treated with 5 µM Lovastatin for 6 h and then utilized to assess expression levels of structural components of caveolae including Cav1, Cav2, Cavin1 (aka PTRF), Cavin2 (aka SDPR), Cavin3 (aka SRBC) and Cavin4 (aka MURC). Cholesterol disruption by Lovastatin resulted in downregulation of each structural component of caveolae. Error bars correspond to SEM from n = 3 technical replicates with statistical significance assessed using two-way ANOVA with Bonferroni correction for multiple comparisons, * p < 0.05, ** p < 0.01, *** p < 0.001.