Differential Expression of Proteins Associated with the Hair Follicle Cycle - Proteomics and Bioinformatics Analyses

Abstract

Hair follicle cycling can be divided into the following three stages: anagen, catagen, and telogen. The molecular signals that orchestrate the follicular transition between phases are still unknown. To better understand the detailed protein networks controlling this process, proteomics and bioinformatics analyses were performed to construct comparative protein profiles of mouse skin at specific time points (0, 8, and 20 days). Ninety-five differentially expressed protein spots were identified by MALDI-TOF/TOF as 44 proteins, which were found to change during hair follicle cycle transition. Proteomics analysis revealed that these changes in protein expression are involved in Ca2+-regulated biological processes, migration, and regulation of signal transduction, among other processes. Subsequently, three proteins were selected to validate the reliability of expression patterns using western blotting. Cluster analysis revealed three expression patterns, and each pattern correlated with specific cell processes that occur during the hair cycle. Furthermore, bioinformatics analysis indicated that the differentially expressed proteins impacted multiple biological networks, after which detailed functional analyses were performed. Taken together, the above data may provide insight into the three stages of mouse hair follicle morphogenesis and provide a solid basis for potential therapeutic molecular targets for this hair disease.

Fig 1. Representative images of skin color, hair regrowth and hematoxylin–eosin (HE) staining of skin samples.

(A) telogen (0 d, pink); (B) anagen (8 d, black); and (C) catagen (20 d); (D-F) Stage-specific morphology of distinct HF compartments, skin thickness, and hair follicle percentage. Scale bar: 100 μm. (D) entire hair follicle resides in the dermis. (E) hair bulb in the subcutis. Tip of the hair shaft emerges through the epidermis. (F) narrower bulb and shorter hair follicle than those in anagen.

Fig 2. Cluster analysis of the expression levels of 44 differentially expressed protein spots in the three phases.

Three clusters (C1, C2, C3) corresponding to three distinct expression patterns were identified. Green represents down-regulated expression, whereas red indicates up-regulated levels.

Fig 3. Potential biological network of differentially expressed proteins during hair cycling.

Cellular processes (yellow squares) involved in each cluster and all relevant proteins (red ovals) validated by literates (the line). Regulation events are displayed with arrows. (A) The cellular processes of high expressed proteins in telogen involved in C1; (B) The cellular processes of high expressed proteins in anagen involved in C2; (C) The cellular processes of high expressed proteins in catagen involved in C3.

Fig 4. The GO enrichment analysis performed by the DAVID bioinformatics resources online.

The results were grouped based on the biological process (BP), molecular function (MF) and cellular component (CC). Statistically significant differences (p-value <0.05) were determined using Fisher’s exact test.

Fig 5. Protein expression confirmation by western blot.

(A) Left panel: Results of Western blot analysis performed with specific protein antibodies and protein extracts from mouse skin. (B) Right panel: Magnified spot images of the same molecular weights distributed in 2-DE gels.