| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 4, 2003 as doi:10.1096/fj.03-0140fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,
* Department of Anatomy,
Department of Cell Biology, Medical School, FIN 33014 University of Tampere,
Department of Urology, and
Department of Clinical Chemistry, Tampere University Hospital, Tampere, Finland
2Correspondence: Department of Anatomy, Medical School, FIN 33014, University of Tampere, Finland. E-mail: loyalo{at}uta.fi
SPECIFIC AIMS
We investigated the biological activities of 25-hydroxyvitamin D3 (25OHD3) and 1
,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in two human prostatic stromal primary cultures.
PRINCIPAL FINDINGS
1. Establishment of the prostatic stromal primary cultures
Two primary cultures, designated P29SN and P32S, derived from a normal area of prostatic carcinoma and adenoma, respectively, were almost exclusively fibroblasts in type and expressed vitamin D receptor.
2. 25OHD3 at physiological concentrations inhibits cell growth
Growth of P29SN cells was significantly inhibited by 250 nM (P<0.01), 1000 nM (P<0.0001) of 25OHD3, and 10 nM of 1,25-(OH)2D3 (P<0.001). Antiproliferative action of 25OHD3 was dose dependent. The growth of P32S cells was not inhibited by 10 nM of 1,25-(OH)2D3 but significantly inhibited by 100 (P<0.01), 250 (P<0.01), and 1000 nM (P<0.0001) of 25OHD3.
3. The prostatic stromal cells possess active 1-hydroxylase
Immunoblotting analysis using an anti-mouse 25-hydroxyvitamin D3-1-hydroxylase (1-hydroxylase, CYP27B1) antibody showed a single band at 56 kDa in stromal primary cultures and a weak band in human prostate cancer cells DU145 and LNCaP. Quantitative real-time RT-PCR showed a detectable and similar level of 1-hydroxylase mRNA in both stromal primary cultures. Only in P29SN cells did 100 nM 25OHD3 at 6 h cause statistically significant up-regulation of 1-hydroxylase mRNA (2-fold, P<0.05). 1-Hydroxylase activity in P29SN cells was 30 fmol·mg protein–1·h–1, which was inhibited by 1000 nM SDZ88-357 (a specific 1-hydroxylase inhibitor) to 7 fmol·mg protein–1·h–1.
4. Expression of 24-hydroxylase mRNA by a physiological concentration of 25OHD3 in stromal primary cultures
Quantitative real-time RT-PCR showed that 1,25-(OH)2D3 at a physiological concentration (0.1 nM) had no effect on expression of 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase, CYP24) mRNA at 6 h in either stromal primary culture whereas 10 nM 1,25-(OH)2D3 markedly increased the mRNA level of 24-hydroxylase in both cultures; induction in P32S cells appeared to be much stronger than that in P29SN cells.
In contrast, 25OHD3 exhibited a dose- and time-dependent induction of 24-hydroxylase mRNA at physiological concentration. 25OHD3 (100 nM) had a small but statistically nonsignificant effect at 6 h in each primary culture; 250 nM enhanced the mRNA level of 24-hydroxylase in P29SN (200-fold at 6 h, P<0.01, Fig. 1
A; 140-fold at 24 h, P<0.05; 90-fold at 48 h, P<0.01), and in P32S (4-fold at 6 h, P<0.05, Fig. 1B
; 7-fold at 24 h, P>0.05; 5-fold at 48 h, P<0.05). 1000 nM 25OHD3 for 6 h caused much higher inductions in P29SN (Fig. 1A
) and P32S (Fig. 1B
) cells, respectively.
|
5. Inhibition of 1-hydroxylation and/or 24-hydroxylation increases the biological activity of 25OHD3
In both primary cultures, the induction of 24-hydroxylase mRNA by 10 nM 1,25-(OH)2D3 was enhanced by a specific inhibitor of 24-hydroxylase, VID400. 25OHD3 (250 nM) with VID400 exhibited an 8-fold (P<0.05) and 60-fold (P<0.05) stimulatory effect compared with 250 nM 25OHD3 alone in P29SN (Fig. 2
A) and P32S cells (Fig. 2B
), respectively. These results suggest there is significant inactivation of 1,25-(OH)2D3 and 25OHD3 by 24-hydroxylase.
|
To investigate whether 25OHD3 is active without 1-hydroxylation, we added a specific inhibitor for 1-hydroxylase, SDZ88-357. When the cells were treated with 250 nM 25OHD3 and 1000 nM SDZ88-357 in combination, 24-hydroxylase mRNA was induced 820-fold in P29SN cells (Fig. 2A
) and 8-fold in P32S cells (Fig. 2B
), indicating that 250 nM 25OHD3 is 4-fold (P>0.05) and 2-fold (P>0.05) more effective than in the absence of 1-hydroxylase inhibitor in P29SN and P32S cells, respectively. When cells were exposed to 250 nM 25OHD3 with 100 nM VID-400 and 1000 nM SDZ88-357 in combination, 24-hydroxylase mRNA was induced 1200-fold in P29SN cells (Fig. 2A
) and 16-fold in P32S cells (Fig. 2B
). These results suggest that 250 nM 25OHD3 is capable of inducing 24-hydroxylase mRNA and 1-hydroxylation is not a prerequisite for its hormonal activity of 25OHD3.
6. 24-Hydroxylase mRNA is induced by 25OHD3 and 1,25-(OH)2D3 in LNCaP cells
To determine whether this phenomenon exists in LNCaP cells, a prostatic epithelial cell line expressing extremely low level of 1-hydroxylase protein, we studied the effect of 25OHD3 and 1,25-(OH)2D3 on 24-hydroxylase mRNA. Both 10 nM 1,25-(OH)2D3 and 1000 nM 25OHD3 time-dependently induced the expression of 24-hydroxylase mRNA. 10 nM 1,25-(OH)2D3 increased 24-hydroxylase mRNA level 1218-fold (P>0.05), 4294-fold (P<0.01), and 2513-fold (P<0.05) at 6, 24, and 48 h, respectively. 1000 nM 25OHD3 increased 24-hydroxylase mRNA level 3730-fold (P<0.05), 11618-fold (P<0.01), and 9300-fold (P<0.05) at 6, 24, and 48 h, respectively. However, neither 1,25-(OH)2D3 (0.1 nM) nor 100–250 nM 25OHD3 affected the expression of 24-hydroxylase mRNA at 24 h in LNCaP cells. These results demonstrate that 1000 nM 25OHD3 specifically induced 24-hydroxylase mRNA expression in LNCaP cells, which are less sensitive to 25OHD3 than primary prostatic stromal cells. 25OHD3 (1000 nM) was more potent in increasing 24-hydroxylase mRNA expression than 10 nM 1,25-(OH)2D3.
CONCLUSIONS AND SIGNIFICANCE
We propose a novel vitamin D3 endocrine system based on the liver hormone 25OHD3, which regulates cell proliferation and gene expression at physiological concentrations in prostatic stromal cells (Fig. 3
). On the other hand, 1,25-(OH)2D3 at physiological concentrations is not an active hormone in the same cells.
|
It has been generally accepted that 1,25-(OH)2D3 is the active form of vitamin D and 25OHD3 is biologically insignificant except for being a precursor. Under physiological conditions, the potency of 1,25-(OH)2D3 in mediating calcium and phosphorus homeostasis is clearly higher than that of 25OHD3. 1,25-(OH)2D3 has been shown to suppress cell proliferation and mediate differentiation only at pharmacological concentrations, which cause the severe side effects of hypercalcemia and hypercalciuria. In contrast, 25OHD3 has a low calcemic effect but a high antiproliferative potency, and is therefore a potent anticancer vitamin D3 metabolite. A serum concentration of 1,25-(OH)2D3 is 1000-fold higher than that of 25OHD3. Therefore, two distinct vitamin D3 endocrine systems have evolved to operate at different sensitivity levels.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0140fje 
This article has been cited by other articles:
|
|
 |
|
  B. Diesel, J. Radermacher, M. Bureik, R. Bernhardt, M. Seifert, J. Reichrath, U. Fischer, and E. Meese Vitamin D3 Metabolism in Human Glioblastoma Multiforme: Functionality of CYP27B1 Splice Variants, Metabolism of Calcidiol, and Effect of Calcitriol Clin. Cancer Res., August 1, 2005; 11(15): 5370 - 5380. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Ren, L. Nguyen, S. Wu, C. Encinas, J. S. Adams, and M. Hewison Alternative Splicing of Vitamin D-24-Hydroxylase: A NOVEL MECHANISM FOR THE REGULATION OF EXTRARENAL 1,25-DIHYDROXYVITAMIN D SYNTHESIS J. Biol. Chem., May 27, 2005; 280(21): 20604 - 20611. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
