Biochemical Engineering Journal | 2021
CRISPR/Cas9-mediated tryptophan hydroxylase 1 knockout decreases calcium transportation in goat mammary epithelial cells
Abstract
Abstract Calcium is one of the major mineral nutrients in goat milk. Tryptophan hydroxylase 1 (TPH1) is a rate-limiting enzyme catalyzing hydroxylation of l -tryptophan into 5-hydroxytryptamine (5-HT, serotonin) essential for maintaining calcium homeostasis. In this study, the TPH1 knockout goat mammary epithelial cells(GMEC) heterozygous clone with no off-target effects were obtained after transfection of the Cas9/sgRNA expression vector firstly. Then the content of 5-HT, intracellular calcium level, and abundance of essential genes related to calcium transportation was evaluated and compared in wild-type GMEC, TPH1 knockout GMEC, to explore the impact of TPH1 on calcium transportation, respectively. Wild-type GMEC and TPH1 knockout GMEC were further treated with exogenous 5-HTP to confirm the role of TPH1 in regulating calcium homeostasis in GMEC. The 5-HT synthesis and intracellular calcium level decreased in TPH1 gene knockout GMEC. The mRNA abundance of secretory-pathway Ca2+-ATPase1 (SPCA1) and plasma membrane Ca2+-ATPase1 (PMCA1) were up-regulated while the mRNA abundance of secretory-pathway Ca2+-ATPase2 (SPCA2) was down-regulated in TPH1 knockout GMEC. Up-regulation of the parathyroid hormone-related peptide (PTHrP), a key regulator of mammary calcium metabolism, induced by 5-HTP were blocked by TPH1 gene knockout. Results suggested that TPH1 knockout decreased calcium transportation via PTHrP and calcium transportation-related factors in GMEC.