1,25(OH)2-vitamin D3 actions on cell proliferation, size, gene expression, and receptor localization, in the HL-1 cardiac myocyte

Abstract

The steroid hormone 1,25(OH)(2)-vitamin D(3) [1,25D] has been shown to affect the growth and proliferation of primary cultures of ventricular myocytes isolated from neonatal rat hearts. The research presented here shows that the vitamin D receptor [VDR] is present in murine cardiac myocytes (HL-1 cells), and that 1,25D affects the growth, proliferation and morphology of these cells. In addition we show that 1,25D effects expression of ANP, myotrophin, and c-myc. Furthermore, 1,25D effects expression and localization of the VDR within the cell. Murine HL-1 cardiac myocytes were grown and treated with 1,25D in culture, and growth and morphology were assessed with microscopic analysis. Cells were counted and protein levels were evaluated through Western blot analysis. Subcellular localization of the VDR was determined using immunofluorescence and confocal microscopy. 1,25D was found to decrease proliferation and alter cellular morphology of the HL-1 cells. Treatment with 1,25D increased expression of myotrophin while decreasing expression of atrial natriuretic peptide [ANP] and c-myc. 1,25D treatment also increased expression and nuclear localization of the VDR in these cardiac myocytes. Thus 1,25D is an important hormone involved in modulating and maintaining heart cell structure and function.

Figure 1. Effect of 1,25D on cell growth

A-B) Cells treated for 3 days with varying concentrations of 1,25D show reduced proliferation and slight hypertrophy relative to control. C-D) Photomicrograph of cells treated for 2 days with (C) control v. (D) 100nM 1,25D.

Figure 2. Western blot of HL-1 gene products

A) Time dependent expression of VDR at 1 hr and 24 hrs when treated with 100nM 1,25D (D₃) v. control (C). B) Dose dependent expression of VDR at 24 hrs when treated with 0.01 to 100 nM 1,25D v. control. C) Expression of VDR, ANP, myotrophin, and c-myc relative to control when treated with 100nM 1,25D for 24 hrs. D) Dose dependent expression of ANP when treated with 0.01 to 100 nM 1,25D v. control.

Figure 3. VDR Ligand Binding Assay in HL-1 Cytosol

HL-1 cells 24 hours post 1:2 split of confluent flask were assayed as previously described [2]. Scatchard transformation of data shown as inset.

Figure 4. Immunofluorescent analysis of VDR

Confocal images of HL-1 cells treated with 100 nM 1,25D (B) v. control (A) for 24 hrs. To ensure specific staining a VDR specific peptide, SC1008P (Santa Cruz), was pre-incubated with primary VDR Ab (C). Adequate Ab penetration and cell architecture preservation is evidenced by actinin staining (D). All digital images are 0.35 um sections captured via Olympus FV500. VDR primary Ab is SC1008 with FITC conjugated secondary AP307F (Chemicon). Actinin primary Ab is A7811 with FITC conjugated secondary A8711 (both Sigma).