Nerve growth factor regulates the proliferation of cashmere goat outer root sheath cells through the activation of cAMP-binding protein

Abstract

The quality and yield of cashmere are closely related to the growth and development of the secondary hair follicles of cashmere goats. Outer root sheath cell (ORSC) proliferation is considered particularly important for the growth and development of hair follicles; however, the factors affecting this are unclear. We previously showed that the expression of nerve growth factor (NGF) in secondary follicle ORSCs was significantly higher in the anagen phase than in the catagen phase, suggesting that NGF promotes ORSC proliferation. To investigate whether ORSCs secrete NGF, and to investigate its role in ORSCs from goat secondary hair follicles, we used enzyme linked immunosorbent assay (ELISA) to show that NGF levels in cultured ORSC medium decreased significantly with the passage of time (P < 0.05). Immunofluorescence analysis detected the transcription factor cAMP-binding protein (CREB) in ORSCs, with a stronger signal intensity observed in the nucleus than in the cytoplasm. An alkaline phosphatase reporter system assay showed that the exogenous addition of 20 ng/mL or 100 ng/mL recombinant NGF protein significantly enhanced the activity of CREB in ORSCs, while treatment with K252α, which inhibits tropomyosin receptor kinase A (TrkA), revealed that NGF enhanced intracellular CREB activity in an NGF/TrkA-dependent manner. Real-time quantitative PCR investigated the expression of proliferating cell nuclear antigen (PCNA) mRNA after treatment with NGF or the CREB inhibitor ICG-001. The NGF/TrkA/CREB system was found to play an important role in maintaining and promoting the expression of PCNA, which will provide a theoretical basis for marker-assisted breeding of cashmere goats.