This study was conducted to investigate the potential mechanisms via which treatment of young goats with MT is able to promote the differentiation and development of secondary hair follicles in their skin. The results show that MT regulates the MAPK pathway in goats by activating the circMPP5/miR-211/MAPK3 axis to promote the differentiation and proliferation of secondary hair follicles, thereby improving the quality and yield of Cashmere. Furthermore, this MT-treatment effect occurred in both the current and subsequent Cashmere growing seasons indicating a potential enhancement of lifetime Cashmere production from these goats.
As in other studies where MT has been used to enhance fibre production in goats and rabbits[23, 24, 25] the present results confirm this is achieved without effect on live weight or growth performance of the animals. As a textile fibre, the quality of Cashmere is improved by reducing fibre diameter and increasing its length[26, 27] and the improvements in these parameters generated here by the treatment with MT has also been recorded in our earlier studies and by others[28, 29]. These effects of MT are achieved by advancing the onset of secondary follicle activity and by increasing the total number of active secondary hair follicles[30, 31] as can be deduced from the various indices of secondary follicle numbers and activity recorded here. Persistence of the fibre growth improvement into the second growth season following MT did not occur in our earlier study[32], so this finding may not be a consistent outcome of the treatment. Likewise, there is no published evidence of the treatment affecting fibre production by offspring of treated animals[33, 34].
Release of endogenous MT from the pineal gland of adult animals is regulated by changes in daily photoperiod[35] which explains why the naturally-occurring changes in fibre production are linked to seasons of the year. However, differentiation and development of hair follicles in goats begins before birth and is largely completed by about 6 months of age, prior to entering the annual hair growth cycle[8, 10, 36]. This explains why treatment of the goats with MT at birth is able to influence the subsequent Cashmere growth cycles. Specific protein markers (biomarkers) of proliferation of secondary hair follicle cells such as keratin 14 (K-14)[37, 38] and Wnt10a[39, 40, 41, 42, 43] and their genes were elevated in the MT group whereas expression and protein levels of fibrinolectin (Fn1), an indicator of cell maturation[44, 45, 46], were lowered.
Whole transcriptome sequencing of extracts from the skin involving epithelial regions where actively growing hair follicles are located[47, 48]may provide markers for breed selection[49] and, importantly for the present study, can reveal gene expression activated by the MT treatment applied in this study, especially those relating to secondary hair follicles[50, 51]. The MAPK signaling pathway is one of the aggregation pathways of cellular information transmission induced by extracellular signals, including extracellular growth factor, extracellular growth factor receptor, and intracellular factors[52, 53]. As well as MT, insulin, platelet growth factor receptor and fibroblast growth factors (FGFs) can affect hair follicle growth by regulating the MAPK signaling pathway[54, 55, 56]. FGF20 controls the formation of secondary hair follicles and there is a high expression of FGF2, FGF21 and FGF-receptor genes during growth of secondary hair follicles[57, 58]. Extracellular signal-regulated kinases (ERKs, also known as MAP kinases) are vital proteins in the differentiation, proliferation and survival of epidermal stem cells[59, 60, 61]. In the present study, genes for the key extracellular and intracellular factors, FGF2, FGF21, FGFR, and MAPK3 (ERK1) in the MAPK pathway showed an up-regulation trend following MT treatment, providing strong evidence that MT exerts its influence on secondary hair follicles via effects on the MAPK pathway.
There is an increasing body of evidence for miRNA having a key role in the early development of hair follicles[62, 63]. Ocu-miR-205 can promote hair follicles from the anagen to catagen stage by regulating the expression of genes for proteins in the notch and bone BMP signaling pathways[64]. Other studies showed that miRNA-203, miRNA-214 and miRNA-195-5p express the genes which regulate the development of hair follicles in Cashmere goats[65, 66] and that MT can influence these miRNAs in the hair follicles of Cashmere goats[67, 68]. miRNA-211 can regulate the MAPK3 pathway negatively, so it is in keeping with its role in this pathway that MT reduced miR-211 in the present study. CircRNA can compete with miRNA to regulate gene expression[69]and is a component of circRNA/miRNA/mRNA involved in regulatory networks as diverse as hepatocellular carcinoma (HCC), contraction of skeletal muscle[70] and proliferation of myoblasts[71]. Differentially expressed circRNAs have been studied in hair follicles of Cashmere goats[72] and Angora rabbits[73]. The targeted binding relationship between miRNA-27b-3p and circRNA3236, miRNA-16b-3p and miR-16b-3p revealed that circRNA regulates gene expression by binding miRNA, thereby controlling hair follicle proliferation and fibre production in Cashmere goats[74]. The present study adds to this body of knowledge by its examination of the differentially expressed circRNAs under the stimulatory influence of MT. The results show that MT in goats can competitively bind miR-211 through circMPP5 thus enhancing expression of the target gene MAPK3 and promoting differentiation of secondary hair follicle cells.