Supplemental Data

The Molecular Basis of Vitamin D Receptor

and β-Catenin Crossregulation Salimuddin Shah, Md Naimul Islam, Sivanesan Dakshanamurthy, Imran Rizvi, Mahadev Rao, Roger Herrell, Glendon Zinser, Meggan Valrance, Ana Aranda, Dino Moras, Anthony Norman, JoEllen Welsh, and Stephen W. Byers Supplemental Experimental Procedures

1,25D mediated repression of β-catenin signaling does not depend on E- or VE-

cadherin: Previous studies have established the ability of 1,25D to repress β-catenin

signaling (Easwaran et al., 1999; Palmer et al., 2001; Shah et al., 2003) (Figure S1A).

Because cadherins can inhibit the signaling activity of β-catenin it is possible that the

repressive effect of 1,25D is indirectly due to increased cadherin expression. To directly

test the role of cadherins in mediating the effects of 1,25D, experiments were performed

with SKBR-3 cells which do not express E-cadherin due to a homozygous deletion of the

gene and make low levels of β-catenin protein. Although retinoic acid increased the

expression of an unknown cadherin in these cells, this increased cadherin expression was

not responsible for retinoic acid-mediated repression of β-catenin signaling (Shah et al.,

2002). 1,25D repressed β-catenin signaling even though SKBR3 cells do not express E-

cadherin (Figure S1B). In contrast to retinoic acid, 1,25D does not change cadherin levels

in SKBR3 cells and demonstrates that 1,25D effects on β-catenin signaling can be

mediated independently of cadherins.

1,25D mediated repression of β-catenin signaling is VDR-dependent: To test the role

of the VDR in mediating the effects of 1,25D on β-catenin signaling we used mammary

tumor cells derived from VDR+/+ and VDR-/- mice and measured VDR (VDRE-luc)

(Figure S1C)and β-catenin (TOPFlash) reporter activities (Figure 3 main text) (Zinser et

al., 2003). Both cell lines expressed similar levels of β-catenin and E-cadherin with or

without 1,25D. As expected, 1,25D treatment increased VDR reporter activity in VDR+/+

cells but not in VDR-/- cells (Figure 2A). Similarly, β-catenin activity was decreased in

VDR+/+ cells after 1,25D treatment but it was unaffected in VDR-/- cells (Figure 3 main

text).

1,25D mediated activation of VDR reporter is attenuated in β-catenin-/- cells: As

shown above, exogenous expression of β-catenin potentiates 1,25D effects on VDR

reporter activity. In addition, activation of VDR reporter by 1,25D is high in SW480 cells

even though they express very low levels of VDR (Figure S2A and C). SW480 cells have

very high levels of β-catenin protein and signaling activity due to mutation in the APC

gene. In contrast, activation of VDR reporter by 1,25D is lower in HEK293 cells even

though they express high levels of VDR protein (Figure S1B and C). HEK293 cells have

relatively low levels of endogenous β-catenin signaling activity (Figure 3C). To formally

test if endogenous β-catenin plays a role in VDR activation by 1,25D we used H28

mesothelioma cells, which have a homozygous deletion of the β-catenin gene (Usami et

al., 2003). H28 cells express significant levels of VDR but are only slightly responsive to

1,25D activation of the VDR reporter (Figure 3 main text and S1C). However, following

transfection of β-catenin, VDR activation by 1,25D was significantly increased

suggesting a role for β-catenin in the activity of VDR (Figure 3 main text). Additionally,

1,25D treatment was able to repress TOPFlash activity in H28 cells transfected with full

length β-catenin (Figure S2D).

Supplemental References

Easwaran,V., Pishvaian,M., Salimuddin, and Byers,S. (1999). Cross-regulation of beta-catenin-LEF/TCF and retinoid signaling pathways. Curr. Biol 9, 1415-1418.

Palmer,H.G., Gonzalez-Sancho,J.M., Espada,J., Berciano,M.T., Puig,I., Baulida,J., Quintanilla,M., Cano,A., de Herreros,A.G., Lafarga,M., and Munoz,A. (2001). Vitamin D(3) promotes the differentiation of colon carcinoma cells by the induction of E-cadherin and the inhibition of beta-catenin signaling. J Cell Biol 154, 369-387.

Shah,S., Hecht,A., Pestell,R., and Byers,S.W. (2003). Trans-repression of beta-catenin activity by nuclear receptors. J Biol Chem 278, 48137-48145.

Shah,S., Pishvaian,M.J., Easwaran,V., Brown,P.H., and Byers,S.W. (2002). The role of cadherin, beta -catenin, and AP-1 in retinoid-regulated carcinoma cell differentiation and proliferation. J. Biol. Chem.

Usami,N., Sekido,Y., Maeda,O., Yamamoto,K., Minna,J.D., Hasegawa,Y., Yoshioka,H., Imaizumi,M., Ueda,Y., Takahashi,M., and Shimokata,K. (2003). Beta-catenin inhibits cell growth of a malignant mesothelioma cell line, NCI-H28, with a 3p21.3 homozygous deletion. Oncogene 22, 7923-7930.

Zinser,G.M., McEleney,K., and Welsh,J. (2003). Characterization of mammary tumor cell lines from wild type and vitamin D3 receptor knockout mice. Mol. Cell Endocrinol. 200, 67-80.

Figure S1: 1,25D repression of β-catenin signaling is VDR dependent and cadherin

independent: (A) SW480 cells were transfected with 150 ng of TCF responsive reporter

(TOPFlash) construct and TCF binding site mutated version of TOPFlash (FOPFlash)

along with renilla luciferase (10ng). 24 hours after transfection, cells were treated with

1µΜ 1,25D for another 24 hours. Luciferase activity was measured as described in

Material and Methods and luminescence values are plotted as ratio of TOPFlash/renilla or

FOPFlash/renilla. (B) As (A) but using cadherin negative β-catenin transfected SKBR-3

cells. (C) VDR+/+ or VDR-/- mouse mammary epithelial cells, were transfected with

VDRE-luciferase and renilla luciferase and treated with 1 µM 1,25D for 24 hours.

A

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Luciferase activity was detected and plotted as relative luminescence units. (C inset)

Western blot of VDR in VDR+/+ and VDR-/- cells.

Figure S2: β-catenin potentiates VDR reporter activity: (A) SW480 cells were

transfected with VDRE-luciferase and renilla luciferase. After 24 hours of transfection,

cells were treated with 1,25D for another 24 hours. (B) Same as (A) with HEK293 cells.

(C) β-catenin and VDR protein expression in SW480, HEK293 and NCI-H28 cells. (D)

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NCI-H28 cells were transfected with β-catenin and TOPFlash and renilla luciferase. 24

hours after transfection cells were treated with 1,25D for another 24 hours and luciferase

activity measured. Data is represented as relative luciferase units.