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Vitamin D reduces hepatic stellate cell proliferation in vitro

Published online by Cambridge University Press:  24 November 2016

P.S. Gibson ,
K. Hart ,
E. Fitzpatrick ,
A. Dhawan ,
S. A. Lanham-New  and
J.B. Moore
P.S. Gibson
Affiliation:
Nutritional Sciences Department, Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7XH
K. Hart
Affiliation:
Nutritional Sciences Department, Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7XH
E. Fitzpatrick
Affiliation:
Paediatric Liver Centre, King's College Hospital, London SE5 9PJ
A. Dhawan
Affiliation:
Paediatric Liver Centre, King's College Hospital, London SE5 9PJ
S. A. Lanham-New
Affiliation:
Nutritional Sciences Department, Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7XH
J.B. Moore
Affiliation:
Nutritional Sciences Department, Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7XH School of Food Science and Nutrition, University of Leeds, Leeds LS2 9JT
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Abstract

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Activated hepatic stellate cells (HSCs) are a key contributor to liver fibrosis(Reference Moreira1) and drive the progression to advanced disease for many liver conditions, including non-alcoholic fatty liver disease. Vitamin D has been shown to have anti-proliferative effects on colorectal cancer cells(Reference Alvarez-Diaz, Valle and Ferrer-Mayorga2); however less is known about its effects on hepatic stellate cells. The aim of these experiments were to determine in vitro: (i) basal protein expression of the vitamin D receptor (VDR); (ii) confirmation of 1α,25(OH)2D3 (1,25-VD3) phenotypic effect; (iii) the time and dose response to 1,25-VD3 treatment.

Three human immortalised cell lines: HT29, colorectal adenocarcinoma; HepG2, hepatocellular carcinoma, and LX-2, hepatic stellate cells, were cultured using standard methods. Several sources of 1,25-VD3 were assessed. VDR protein expression was analysed by immunoblotting. VDR and CYP24A1 mRNA expression was measured in LX-2 and HepG2 cells at 0, 1, 2, 4, 6, 8, 10, 12 and 24 hour time points after treatment with 10nM of 1,25-VD3. LX-2, HepG2 and HT29 were treated with a range of 1,25-VD3 concentrations (1μg-1 ng) and cell proliferation was measured by clonogenic assay using crystal violet staining.

Untreated LX-2 cells had a higher abundance of VDR protein than HepG2 cells (Figure 1A). Correspondingly, basal VDR mRNA expression was higher in LX-2 in comparison to HepG2 cells (P < 0·0001). However, mRNA expression for CYP24A1 was much lower in LX-2 compared to HepG2 cells (P < 0·0001). Treatment with 1,25-VD3 dramatically reduced hepatic stellate cell proliferation; a dose-dependent response was observed with 1μM and 100nM of 1,25-VD3 eliciting a significant reduction in cell colonies (P = 0·0005 and P = 0·016 respectively; n 4; Figure 1B) corresponding to 95 % and 82 % fewer colonies. The same trend was seen in HT29 cells (P = 0·053; n 3; Figure 1C).

Fig. 1.

Basal levels of VDR in LX-2 and HepG2 cells (A). Cell proliferation of LX-2 (B) and HT29 (C) cells in response to 1,25-VD3. Data presented as mean + SEM. V: vehicle; VDR: vitamin D receptor.

Unexpectedly, our initial 1,25-VD3 treatment appeared to have no effect on VDR and CYP24A1 mRNA expression. After an alternative vitamin D was sourced, a clear reduction in cell proliferation in response to 1,25-VD3 was observed in both LX-2 and HT29 cells. Future experiments will determine the associated transcriptional response in co-treatment with lipid loading.

References

1.Moreira, RK (2007) Arch Pathol Lab Med 131: 1728–1734Google Scholar
2.Alvarez-Diaz, S, Valle, N, Ferrer-Mayorga, G et al. (2012) Hum Mol Genet 21 (20): 2157–2165Google Scholar
Figure 0

Fig. 1. Basal levels of VDR in LX-2 and HepG2 cells (A). Cell proliferation of LX-2 (B) and HT29 (C) cells in response to 1,25-VD3. Data presented as mean + SEM. V: vehicle; VDR: vitamin D receptor.