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Effect of all-trans retinoic acid on casein and fatty acid synthesis in MAC-T cells.

ABSTRACT: OBJECTIVE:Caseins and fatty acids of milk are synthesized and secreted by the epithelial cells of the mammary gland. All-trans retinoic acid (ATRA), an active metabolite of vitamin A, has been shown to promote mammary development. This study was conducted to determine the effect of ATRA on casein synthesis and fatty acid composition in MAC-T cells. METHODS:MAC-T cells were allowed to differentiate for 4 d, treated with ATRA (0, 1.0, 1.5, and 2.0 ?M), and incubated for 3 d. We analyzed the fatty acid composition, the mRNA expression of casein and fatty acid synthesis-related genes, and the phosphorylation of casein synthesis-related proteins of MAC-T cells by gas chromatography, quantitative polymerase chain reaction, and western blotting, respectively. RESULTS:In MAC-T cells, ATRA increased the mRNA levels of ?S1-casein and ?-casein, janus kinase 2 (JAK2) and E74-like factor 5 of the signal transducer and activator of transcription 5 ? (STAT5-?) pathway, ribosomal protein S6 kinase beta-1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 of the mammalian target of rapamycin (mTOR) pathway, inhibited the mRNA expression of phosphoinositide 3-kinase and eukaryotic initiation factor 4E of the mTOR pathway, and promoted the phosphorylation of STAT5-? and S6K1 proteins. Additionally, ATRA increased the de novo synthesis of fatty acids, reduced the content of long-chain fatty acids, the ratio of monounsaturated fatty acids to saturated fatty acids (SFA), the ratio of polyunsaturated fatty acids (PUFA) to SFA, and the ratio of ?-6 to ?-3 PUFA. The mRNA levels of acetyl-CoA carboxylase 1, fatty acid synthase, lipoprotein lipase, stearoyl-CoA desaturase, peroxisome proliferator-activated receptor gamma, and sterol regulatory element-binding protein 1 (SREBP1) were enhanced by ATRA. CONCLUSION:ATRA promotes the synthesis of casein by regulating JAK2/STAT5 pathway and downstream mTOR signaling pathway, and it improves the fatty acid composition of MAC-T cells by regulating SREBP1-related genes.

SUBMITTER: Liao XD

PROVIDER: S-EPMC7206372 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Effect of all-trans retinoic acid on casein and fatty acid synthesis in MAC-T cells.

Liao Xian-Dong XD Zhou Chang-Hai CH Zhang Jing J Shen Jing-Lin JL Wang Ya-Jing YJ Jin Yong-Cheng YC Li Sheng-Li SL

Asian-Australasian journal of animal sciences 20190823 6

<h4>Objective</h4>Caseins and fatty acids of milk are synthesized and secreted by the epithelial cells of the mammary gland. All-trans retinoic acid (ATRA), an active metabolite of vitamin A, has been shown to promote mammary development. This study was conducted to determine the effect of ATRA on casein synthesis and fatty acid composition in MAC-T cells.<h4>Methods</h4>MAC-T cells were allowed to differentiate for 4 d, treated with ATRA (0, 1.0, 1.5, and 2.0 μM), and incubated for 3 d. We anal ...[more]

PMID: 31480153

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Project description:BACKGROUND: Our recent results show that all-trans retinoic acid (ATRA), an active metabolite of vitamin A, induces COX-dependent hyperalgesia and allodynia in rats. This effect was mediated by retinoic acid receptors (RARs) and was associated with increased COX-2 expression in the spinal cord. Since ATRA also up-regulated COX-2 expression in SH-SY5Y human neuroblastoma cells, the current study was undertaken to analyze in these cells the mechanism through which ATRA increases COX activity. METHODS: Cultured SH-SY5Y neuroblastoma cells were treated with ATRA. COX expression and kinase activity were analyzed by western blot. Transcriptional mechanisms were analyzed by RT-PCR and promoter assays. Pharmacological inhibitors of kinase activity and pan-antagonists of RAR or RXR were used to assess the relevance of these signaling pathways. Production of prostaglandin E2 (PGE2) was quantified by enzyme immunoabsorbent assay. Statistical significance between individual groups was tested using the non-parametric unpaired Mann-Whitney U test. RESULTS: ATRA induced a significant increase of COX-2 expression in a dose- and time-dependent manner in SH-SY5Y human neuroblastoma cells, while COX-1 expression remained unchanged. Morphological features of differentiation were not observed in ATRA-treated cells. Up-regulation of COX-2 protein expression was followed by increased production of PGE2. ATRA also up-regulated COX-2 mRNA expression and increased the activity of a human COX-2 promoter construct. We next explored the participation of RARs and mitogen-activated peptide kinases (MAPK). Pre-incubation of SH-SY5Y human neuroblastoma cells with either RAR-pan-antagonist LE540 or MAP kinase kinase 1 (MEK-1) inhibitor PD98059 resulted in the abolition of ATRA-induced COX-2 promoter activity, COX-2 protein expression and PGE2 production whereas the retinoid X receptor pan-antagonist HX531, the p38 MAPK inhibitor SB203580 or the c-Jun kinase inhibitor SP600125 did not have any effect. The increase in RAR-beta expression and extracellular-regulated kinase 1/2(ERK1/2) phosphorylation in ATRA-incubated cells suggested that RARs and ERK1/2 were in fact activated by ATRA in SH-SY5Y human neuroblastoma cells. CONCLUSION: These results highlight the importance of RAR-dependent and kinase-dependent mechanisms for ATRA-induced COX-2 expression and activity.

Project description:1. all-trans-Retinoic acid at concentrations greater than 10(-7)m stimulated the incorporation of d-[(3)H]glucosamine into 8m-urea/5% (w/v) sodium dodecyl sulphate extracts of 1m-CaCl(2)-separated epidermis from pig ear skin slices cultured for 18h. The incorporation of (35)SO(4) (2-), l-[(14)C]fucose and U-(14)C-labelled l-amino acids was not significantly affected. 2. Electrophoresis of the solubilized epidermis showed increased incorporation of d-[(3)H]glucosamine into a high-molecular-weight glycosaminoglycan-containing peak when skin slices were cultured in the presence of 10(-5)m-all-trans-retinoic acid. The labelling of other epidermal components with d-[(3)H]glucosamine, (35)SO(4) (2-), l-[(14)C]fucose and U-(14)C-labelled l-amino acids was not significantly affected by 10(-5)m-all-trans-retinoic acid. 3. Trypsinization dispersed the epidermal cells and released 75-85% of the total d-[(3)H]glucosamine-labelled material in the glycosaminoglycan peak. Thus most of this material was extracellular in both control and 10(-5)m-all-trans-retinoic acid-treated epidermis. 4. Increased labelling of extracellular epidermal glycosaminoglycans was also observed when human skin slices were treated with all-trans-retinoic acid, indicating a similar mechanism in both tissues. Increased labelling was also found when the epidermis was cultured in the absence of the dermis, suggesting a direct effect of all-trans-retinoic acid on the epidermis. 5. Increased incorporation of d-[(3)H]-glucosamine into extracellular epidermal glycosaminoglycans in 10(-5)m-all-trans-retinoic acid-treated skin slices was apparent after 4-8h in culture and continued up to 48h. all-trans-Retinoic acid (10(-5)m) did not affect the rate of degradation of this material in cultures ;chased' with 5mm-unlabelled glucosamine after 4 or 18h. 6. Cellulose acetate electrophoresis at pH7.2 revealed that hyaluronic acid was the major labelled glycosaminoglycan (80-90%) in both control and 10(-5)m-all-trans-retinoic acid-treated epidermis. 7. The labelling of epidermal plasma membranes isolated from d-[(3)H]glucosamine-labelled skin slices by sucrose density gradient centrifugation was similar in control and 10(-5)m-all-trans-retinoic acid-treated tissue. 8. The results indicate that increased synthesis of mainly extracellular glycosaminoglycans (largely hyaluronic acid) may be the first response of the epidermis to excess all-trans-retinoic acid.

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Effect of all-trans retinoic acid on casein and fatty acid synthesis in MAC-T cells.

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Effect of all-trans retinoic acid on casein and fatty acid synthesis in MAC-T cells.

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