Disrupted cholesterol biosynthesis and hair follicle stem cell impairment in the onset of alopecia

Abstract

Background: Hair follicle cycle and the functioning of stem cells in alopecia are influenced by the suppression of cholesterol synthesis and the accumulation of sterol intermediates.

Objective: This investigation aims to elucidate the regulatory function of disrupted cholesterol homeostasis in the functioning of hair follicle stem cells (HFSCs) and the cycling of hair follicles. Additionally, it seeks to provide an understanding of the fate of stem cells in primary cicatricial alopecia (PCA).

Methods: To evaluate the influence of cholesterol on the functionality of hair follicles, a study was conducted to analyse gene expression and pathways associated with hair follicle stem cell markers in scalp samples affected by PCA (LPP, FFA, CCCA, DC, DF, TF). To assess the influence of disrupted cholesterol homeostasis on HFSCs, we conducted experiments involving the administration of 7-dehydrocholesterol (7DHC) and BM15766 (Pharmacological inhibitor of cholesterol biosynthesis), to Human Hair Follicle Outer Root Sheath Cells (HORSCs), as well as C57BL/6 mice, and hair follicle organoid cultures. The study utilised reverse transcription polymerase chain reaction (RT-PCR) to assess gene expression, while immunofluorescence was employed to analyse protein expression. The tracking of stem cell fate was accomplished through the utilisation of a BrdU pulse-chase experiment, while the verification of apoptotic consequences was established by utilising the TUNEL assay. A statistical analysis was conducted to assess the statistical significance of the data.

Results: There was a notable decrease in the expression of HFSC marker genes among patients afflicted with PCA. In vitro data further confirmed the cholesterol inhibition and sterol intermediate accumulation in stem cells, leading to stem cell characteristics' disruption. The experimental group of mice exposed to 7DHC and BM15766 also exhibited a notable inability to initiate hair regrowth. Consequently, this deficiency in hair regrowth resulted in the activation of apoptosis, specifically in the stem cells. Additionally, our confirmatory analysis, which was performed utilising organoid culture, consistently yielded comparable results. The results as mentioned above emphasise the significant importance of cholesterol production in preserving the integrity and functionality of HFSCs, hence providing novel insights into the progression of alopecia.

Conclusion: Individuals with aberrant cholesterol production, especially those affected by PCA, endure a lasting hindrance in hair regrowth stemming from the irreversible damage to their hair follicles. The observed phenomenon is hypothesized to result from the depletion of hair follicle stem cells. Our study reveals further insights that unveil the previously unrecognized role of sterol intermediates in governing hair follicle cycling and stem cell function in PCA. The modulation of cholesterol production and the accumulation of sterol intermediates significantly influence the hair follicle cycle and the role of stem cells in alopecia.

Fig 1. Down -regulated expression of HFSC marker genes in PCA.

Genes directly and indirectly connected with (A) LGR5 and (B) SOX9 are down-regulated. Nodes, genes; green nodes, genes down-regulated in pathway; pink nodes, genes up regulated. Line between two nodes represents the corresponding protein products of the genes can physically interact (according to literature). The difference in intensity reflecting the degree of change in the expression of differentially expressed genes in our database. The difference in intensity reflecting the degree of change in the expression of differentially expressed genes in our database. Compared with the unaffected tissue, (C) SOX 9 gene expression was significantly down-regulated in all PCA subtypes DF, DC, LPP, CCCA but not in TF and FFA. The un paired t-test used for statistical analysis. (D) Real time PCR validation of LGR5 gene expression in unaffected and affected skin in the PCA subtypes TF, DF, DC, FFA, LPP, CCCA (*p < 0.05, **p < 0.01). Compared with the unaffected tissue, LGR5 gene expression was significantly down-regulated in all PCA samples. The un paired t-test used for statistical analysis. (E) Real time PCR validation of SHH gene expression in unaffected and affected skin in the PCA subtypes TF, DF, DC, FFA, LPP, CCCA (*p < 0.05, **p < 0.01). Compared with the unaffected tissue, SHH gene expression was significantly down-regulated in all PCA subtypes DF, DC, FFA, LPP, CCCA but not in TF. The un paired t-test used for statistical analysis. (F) Real time PCR validation of WNT5A gene expression in unaffected and affected skin in the PCA subtypes TF, DF, DC, FFA, LPP, CCCA (*p < 0.05, **p < 0.01). Compared with the unaffected tissue, WNT 5A gene expression was significantly downregulated in all PCA subtypes TF, DC, FFA, LPP, CCCA but not in DF. The one way ANOVA was used for the statistical analysis.

Fig 2. Inhibited cholesterol biosynthesis downregulates hair follicle stem cell marker genes in in vitro model system.

The real-time PCR validation of (A) SOX9 (B) LGR5 (C) WNT 5A (D) SHH gene expression in 7-DHC and BM15766- treated samples, HFORSCs (*p < 0.05, **p < 0.01) is shown. Compared with the controls, treated samples show significant down-regulation in the expression of SOX9 and LGR5. The expression of WNT5A, down-regulated in 7DHC. SHH expressions are down-regulated in BM15766 treated Sample but not in 7-DHC. The one way ANOVA was used for the statistical analysis. Treatment with 7-DHC and BM15766 can reduce the expression of some or all of these genes.. Immunofluorescence analysis showed that (E) SOX9, (F) LGR5 and (G) Wnt 5A (Red) expressions are down-regulated compared with the control. Staining for the merge of SOX9, LGR5 and WNT 5A with DAPI respectively.

Fig 3. 7DHC and BM15766 treated mice (C57BL/6; n = 5) failed to regrow the hairs.

Treatment with topical (A) 7DHC and (B) BM15766 resulted in a reduction in hair growth when compared to mice treated with Ethanol/DMSO. However, while hair growth was successfully restored in the mice treated with the vehicle, it did not recover in the treated group. Real-time PCR validation of LGR5, WNT 5A, SOX9 and SHH gene expression in mouse skin treated with (C) 7DHC and (D) BM15766 compared with vehicle-treated (Ethanol/ DMSO) controls (n =  5; * p,0.05, **p,0.01). Treatment group can down-regulate the expression of some or all of these genes. (E) Labeling with BrdU during the pulse phase and the subsequent chase period, the experiment did not yield conclusive evidence of stem cell populations retaining the BrdU label in 7DHC and BM15766 treated mice. In contrast the control (Ethanol/ DMSO) tissues the experiment showed the presence of BrdU labeling. TMR red imaging revealed a higher number of apoptotic or DNA damaged (red color) cells in the (F) BM15766 and (G) 7DHC treated samples compared to the + ve and vehicle (Ethanol/ DMSO) control group. Tissue counterstained with DAPI(nucleus).

Fig 4. Inhibitory effect of 7DHC and BM15766 in HF organoid culture.

(A)The diagram provides a visual representation depicting the detrimental effects of BM15766 and 7DHC on the hair follicle bulge. The hair follicle bulge, a critical region housing stem cells. The detrimental impact on the bulge region is indicated by structural alterations, disruption of cellular organization, deterioration of hair matrix and the hair fiber pushed upwards within the follicle. The real-time PCR validation of (B) LGR5 and (C) SOX9 gene expression in 7-DHC and BM15766- treated samples, HF (*p < 0.05, **p < 0.01) is shown. Compared with the controls, treated samples show significant down-regulation in the expression of SOX9 and LGR5 The one way ANOVA was used for the statistical analysis. (D) Immunofluorescence imaging of the LGR5 (Alexa fluor 488) in HFs with DAPI counter staining. Compared with the control, treated samples shows reduced expression of LGR5. (E) Immunofluorescence imaging of the expression of K15 (Alexa fluor 594) and TUNEL positive cells, of HFs treated with 7DHC and BM15766. The HFs are counter stained with DAPI and finally merged. The treatment group showed higher apoptotic cells than the control group. Similarly the expression of K15 was drastically reduced when the HFs are exposed to 7DHC and BM15766 (F). TEM analysis of hair follicle (A) Control, (B) Vehicle, (C) BM15766, (D) 7DHC. In (A) control and (B) vehicle control, the sample shows a well-organized structure typical of a hair matrix. Connective tissue sheath that surrounds and supports the matrix cells. Keratin filaments, melanin granules and melanosomes are visible. Minimal intercellular space suggests tight packing of cells, characteristic of the proliferative zone in the hair matrix. While in (C) BM15766 and (D) 7DHC treated samples the matrix cells ruptured and discontinuous. Melanin granules are sparsely distributed and appear partially degraded. The keratin filaments are fragmented and disorganized. Degraded connective tissue sheath. Matrix (Mx), Keratin filaments (KF), Melanin granules (MG), melanosomes (M), Connective tissue sheath (CTS).

Fig 5. In individuals affected by PCA, there is a significant downregulation of genes associated with the cholesterol biosynthetic pathway.

This down-regulation results in the accumulation of sterol intermediates, which triggers inflammatory responses within hair follicle (HF) stem cells, ultimately leading to the apoptosis of these stem cells. The inflammatory reactions involve various elements, such as Toll-Like Receptors (TLR), Interferons (IFN), and Transforming Growth Factor-beta (TGF-β). These inflammatory factors collectively have a suppressive effect on the phosphorylation of SOX9, a crucial protein in stem cell proliferation, particularly in the bulge of the Outer Root Sheath (ORS). This suppression further contributes to damage to the stem cells. To understand the mechanistic aspects of this process, we introduce two key compounds, 7-Dehydrocholesterol (7DHC) and BM15766, which artificially alter the cholesterol biosynthetic pathway. These compounds effectively mimic the conditions experienced by PCA-affected individuals. In this altered state of the cholesterol biosynthetic pathway, we find a significant connection with critical stem cell markers, specifically Hh, WNT, LGR5. The N-terminus of Hh is modified by adding palmitate to a conserved cysteine residue, while the C-terminus is modified with cholesterol. Similarly, WNT proteins undergo palmitoylation at their first conserved cysteine. These lipid modifications are crucial for the precise localization of both WNT and Hh proteins and further effective functioning in the development and cycling of hair follicles.In stem cell regeneration, Hh plays a pivotal role in promoting the proliferation of quiescent stem cells, ultimately leading stem cell regeneration. Similarly, WNT signaling is instrumental in regenerating Hair Follicle Stem Cells (HFSCs) during hair growth.The absence of cholesterol modification in Hh and WNT due to the altered cholesterol biosynthetic pathway disrupts stem cell proliferation. Moreover, this disruption affects downstream targets of the WNT pathway, such as LGR5, leading to the failure of its expression in the lower bulge, ORS, and IRS regions. Coupled with the presence of inflammatory cells, this disruption can culminate in the apoptosis of stem cells, ultimately contributing to the pathology observed in PCA-affected individuals.