Neuropeptides regulate embryonic salivary gland branching through the FGF/FGFR pathway in aging klotho-deficient mice.

Salivary gland branching morphogenesis is regulated by the functional integration of neuronal signaling, but the underlying mechanisms are not fully understood in aging accelerated klotho-deficient (Kl) mice. Here, we investigated whether the neuropeptides substance P (SP) and neuropeptide Y (NPY) affect the branching morphogenesis of embryonic salivary glands in aging Kl mice. In the salivary glands of embryonic Kl mice, morphological analysis and immunostaining revealed that epithelial bud formation, neuronal cell proliferation/differentiation, and the expression of the salivary gland functional marker ZO-1 were decreased in embryonic ductal cells. Incubation with SP/NPY at E12-E13d promoted branching morphogenesis, parasympathetic innervation, and epithelial proliferation in salivary glands of embryonic Kl mice. The ERK inhibitor U0126 specifically inhibited neuronal substance-induced epithelial bud formation in the embryonic salivary gland. RNA-seq profiling analysis revealed that the expression of fibroblast growth factors/fibroblast growth factors (FGFs/FGFRs) and their receptors was significantly regulated by SP/NPY treatment in the embryonic salivary gland (E15). The FGFR inhibitor BGJ389 inhibited new branching formation induced by SP and NPY treatment and ERK1/2 expression. These results showed that aging may affect virtually the development of salivary gland by neuronal dysfunction. The neuropeptides SP/NPY induced embryonic salivary gland development through FGF/FGFR/ERK1/2-mediated signaling.