Ann Dermatol.  2019 Oct;31(5):511-517. 10.5021/ad.2019.31.5.511.

Expression Pattern and Role of Klotho in Human Hair Follicles

• Jin XH
• Pi LQ
• Lee WS

• KMID: 2456227
• DOI: http://doi.org/10.5021/ad.2019.31.5.511

Abstract

BACKGROUND
Klotho protein plays a pivotal role in aging regulation. However, it is unclear whether klotho is expressed in human hair follicles and is correlated with hair growth.
OBJECTIVE
The purpose of this study was to determine the expression pattern and role of klotho in human hair follicles.
METHODS
We examined the klotho expression patterns in human hair follicles from young and aged donors. Furthermore, we examined the functional roles of klotho on human hair growth using klotho siRNA and klotho recombinant protein.
RESULTS
Interestingly, klotho was expressed in human hair follicles at both gene and protein levels. In hair follicles, prominent klotho expression was mainly observed in the outermost regions of the outer root sheath and hair bulb matrix cells. Quantification of klotho protein expression in young and aged donors showed that klotho expression decreased with aging. In human hair follicle organ culture, klotho silencing promoted premature catagen induction and inhibited human hair growth. Otherwise, klotho protein prolonged human hair growth.
CONCLUSION
These results indicate that klotho might be an important regulatory factor for human hair growth and hair cycle change.

Keyword

Hair growth; Human hair follicle; Klotho

MeSH Terms

Aging
Hair Follicle*
Hair*
Humans*
Organ Culture Techniques
RNA, Small Interfering
Tissue Donors
RNA, Small Interfering

Figure

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  Fig. 1 Expression of klotho mRNA and protein in human hair follicles. (A) Reverse transcription polymerase chain reaction was performed on isolated anagen VI stage hair follicles using a specific klotho primer pair. Human kidney tissue was used as a positive control and PBS (no cDNA) was used as a negative control. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. (B) Western blot analysis was performed on isolated anagen VI stage hair follicles using a specific klotho antibody. Human kidney tissue was used as a positive control. β-actin was used as a loading control.

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  Fig. 2 Immunohistochemical localization of klotho in human scalp skin. Paraffin sections of human scalp skin (A~G) and human kidney tissue (H) were stained with anti-klotho antibody. Close-up images of boxed regions of A~H were also shown in A′~H′. Negative control was performed with normal rabbit immunoglobulin G instead of primary antibody in the sections of human hair follicle units (B and B′; G and G′). Positive control was performed with anti-klotho antibody in the sections of human kidney tissue (H and H′). EP: epidermis, SB: sebaceous gland, SG: sweat gland, ORS: outer root sheath, IRS: inner root sheath, Ma: hair matrix, DCT: distal convoluted tubules. Scale bar=100 µm.

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  Fig. 3 Aging dependent expression pattern of klotho in hair follicles. (A) Western blot analysis was performed on individual human hair follicles (aged form 21 to 70). β-actin was used as a loading control. (B) Paraffin sections of young (28 years) and old (65 years) human hair follicle were stained with anti-klotho antibody (a~d). Close up images of boxed regions of a~d were also shown in a′~d′. a and a′, Klotho immunoreactivity in the epidermis from young subject. b and b′, Klotho immunoreactivity in the epidermis from aged subject. c and c′, Klotho immunoreactivity in the outer root sheath from young subject. d and d′, Klotho immunoreactivity in the outer root sheath from aged subject. Scale bar=100 µm.

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  Fig. 4 Effects of klotho silencing on human hair growth and hair cycling. Hair follicles were transfected with klotho siRNA (200 nM) or non-targeting control siRNA. (A) The length of hair follicle unit was measured every day until 3 days. (B) Measurement of hair cycle staging of each hair follicle (anagen, early catagen, mid catagen, late catagen). (C) Calculation of hair cycle score of each hair follicle. (D) Representative images of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells (green) in control or klotho siRNA treated hair follicles. (E) Quantification of cell apoptosis. (F) Representative images of Ki-67 positive cells (green) control or klotho siRNA treated hair follicles. (G) Quantification of proliferative cell. Data are based on 40~50 hair follicle units per group, and are presented as the mean±standard deviation. *p<0.05. Scale bar=100 µm. PI: propidium iodide.

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  Fig. 5 Effects of klotho protein on human hair growth and cycling. Hair follicles were treated with klotho protein (0.1 or 0.5 µg/ml) every other day. Hair follicle organ culture was performed over 8 days. (A) The length of hair follicle unit was measured every other day until 8 days. (B) Measurement of hair cycle staging of each hair follicle (anagen, early catagen, mid catagen, late catagen). (C) Calculation of hair cycle score of each hair follicle. Data are based on 40~50 hair follicle units per group, and are presented as the mean±standard deviation. *p<0.05.

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