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.
Highlights
Calcium is one of the major mineral nutrients in dairy products
The objective of this study is to evaluate the effect of Tryptophan hydroxylase1 (TPH1) knockout through clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology on 5-HT synthesis, intracellular calcium content, expression levels of local calcium regulator and relative abundance of genes-related to calcium transportation
Through 2D and 3D intracellular calcium imaging analysis, calcium content in goat mammary epithelial cells (GMEC) decreased approximately 50% by TPH1 knockout,and this result was similar to the result of TPH1 knockout induced less bone resorption of mice [41]. These results suggested that both 5-HT synthesis and calcium metabolism in GMEC were negatively affected in the TPH1 knockout GMEC model
Summary
Calcium is one of the major mineral nutrients in dairy products. A large amount of calcium was transported from plasma to mammary epithelial cells[4]. The mammary gland generates a large transepithelial calcium gradient in favour of milk[5]. The plasma membrane calcium efflux pumps PMCAs (plasma membrane Ca2+-ATPases), located on the apical membrane, was responsible for the transportation of Calcium from the epithelial cell to milk[6]. Pathways and mechanisms which underlie the movement of calcium across the mammary gland are still worth studying. Calcium is one of the major mineral nutrients in goat milk. Tryptophan hydroxylase (TPH1) is a rate-limiting enzyme catalyzing hydroxylation of L-tryptophan into 5-hydroxytryptamine (5-HT) essential for maintaining calcium homeostasis. The function of TPH1 and 5-HT in goat mammary calcium homeostasis is not well known
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
