Journal of Steroid Biochemistry & Molecular Biology 103 (2007) 416–419

Heart extracellular matrix gene expression profile in the

vitamin D receptor knockout mice
Ayesha Rahman a , Stephen Hershey a , Salahuddin Ahmed b ,
Karl Nibbelink a , Robert U. Simpson a,∗
a Department of Pharmacology, University of Michigan Medical School, 1301 MSRB III, 1150 West Medical Center Drive,
Ann Arbor, MI 48109-0632, United States
b Department of Internal Medicine/Division of Rheumatology, University of Michigan Medical School, BSRB Room 4388,

109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, United States

Abstract

1␣,25-Dihydroxyvitamin D3 [1,25D] deficiency and vitamin D receptor [VDR] genotypes are risk factors for several diseases and disorders
including heart diseases. Extracellular matrix (ECM) remodeling mediated by matrix metalloproteinases [MMPs] contributes to progressive
left ventricular remodeling, dilation, and heart failure. In the present study, we used high-density oligonucleotide microarray to examine gene
expression profile in wild type [WT] and vitamin D receptor knockout mice (VDR KO) which was further validated by RT-PCR. Microarray
analysis revealed tissue inhibitors of metalloproteinases [TIMP-1 and TIMP-3] were significantly under expressed in VDR KO mice as
compared to WT mice which was further validated by RT-PCR. Zymography and RT-PCR showed that MMP-2 and MMP-9 were up regulated
in VDR KO mice. In addition, cross-sectional diameter and longitudinal width of the VDR KO heart myofibrils showed highly significant
cellular hypertrophy. Trichrome staining showed marked increase in fibrotic lesions in the VDR KO mice. Heart weight to body weight ratio
showed ∼41% increase in VDR KO mice when compared to WT mice. This data supports a role for 1,25D in heart ECM metabolism and
suggests that MMPs and TIMPs expression may be modulated by vitamin D.
© 2007 Published by Elsevier Ltd.

Keywords: Vitamin D receptor; Matrix metalloproteinases; Gene expression; Heart

1. Introduction fibrosis, and regulates proliferation and hypertrophy in rat

The biologically active form of vitamin D3 , 1,25D has
a broad range of physiological effects [1,2]. This hormone
functions through the vitamin D receptor (VDR), a member
of the nuclear hormone receptor family, to regulate transcrip-
tion of target genes. In addition to modulating rates of gene
expression, 1,25D also mediates non-genomic and more rapid
effects which are absent in cells isolated from VDR-null mice
[3,4].
Previous studies from our and other labs have shown that
1,25D receptor exists in the rat and human heart [5,6] and
vitamin D3 deficiency increases myocardial contractility and
∗ Corresponding author. Tel.: +1 734 763 3255; fax: +1 734 763 4450.
E-mail address: robsim@umich.edu (R.U. Simpson).
cardiomyocytes [7]. Activation of MMP is known to con-
tribute to the degradation of extracellular matrix (ECM),
myocardial wall thinning, and left ventricular (LV) dilation
[8]. Dysregulated MMP/TIMP production has been demon-
strated in the failing heart [9]. It has also been reported that
collagen structure and functional properties are altered in
cardiomyopathies [10].
In many respects, 1,25D signaling is ideally suited to
genomic analysis because VDR is a direct regulator of gene
transcription with a well-characterized binding site. In the
present study, using microarray analysis and RT-PCR tech-
nique, we studied gene regulation in WT and VDR KO
mice. Our results provide insights into the range of molecular
genetic events underlying the broad physiological actions of
1,25D, including its effects on MMP regulation and collagen
formation in heart.

0960-0760/$ – see front matter © 2007 Published by Elsevier Ltd.

doi:10.1016/j.jsbmb.2006.12.081
 A. Rahman et al. / Journal of Steroid Biochemistry & Molecular Biology 103 (2007) 416–419 417

2. Materials and methods 2.4. Reverse transcriptase polymerase chain reaction

2.1. Animal maintenance To validate microarray results, RT-PCR was done on

All the studies were approved by the institutional animal
care committee at University of Michigan. A breeding colony
of VDR HET mice (+/−) was established from three mice
generously provided by Dr. Marie Demay (Harvard Med-
ical School, Boston, MA). All VDR KO (−/−) and WT
mice (+/+) were fed a diet containing 2% calcium, 1.25%
phosphate and 20% lactose with 2.2 IU vitamin D3 /g (Teklad
Diet #TD.96348, Madison, WI). At 12 months, animals were
weighed, heparnized (1500 U/kg, i.p.) then after 20 min anes-
thetized with Nembutal (162.5 U/kg, i.p.). The heart was
rapidly excised, blotted dry, weighed and the left ventricle
isolated and frozen in liquid nitrogen.
2.2. H&E and trichrome staining
Collagen staining/localization and cardiac muscle fiber
size measurements were done by the Pathology Department
of University of Michigan.
2.3. Gene expression analysis by ECM-specific
microarray
To examine ECM genes in left ventricles of VDR KO and
WT mice, Oligo GEArray ECM gene expression array system
from SuperArray Bioscience Corporation, Bethesda, MD was
used. The mRNA was isolated using TRIzol Reagent (Invitro-
gen. Carlsbad, CA) according to manufacturer’s instructions.
Minimal value background corrected signals were normal-
ized to the averaged intensity of signals.
cDNA synthesized from 5 ␮g of total RNA using the first-
strand cDNA synthesis kit from SuperArray. Primers and
probes for mouse MMP-2, MMP-9, TIMP-1, TIMP-3, and
the housekeeping gene GAPDH were purchased and pro-
cessed per protocol by Superarray.
2.5. Gelatin zymography
Left ventricles (∼50 mg) were homogenized in buffer
(10 mM HEPES, pH 7.5, 150 mM NaCl, 0.2 mM EDTA,
25% glycerol, 100 ␮g/ml PMSF, and aprotinin). Equal pro-
tein (10 ␮g) from each sample was utilized to study MMP-2
and MMP-9 activity using gelatin zymography.
2.6. Statistical analysis
Data were obtained from three samples per experiment
and are presented as mean ± S.E.M. The statistical analysis
was performed using the Student’s t-test.
3. Results
3.1. Increased heart/body weight ratio in VDR KO mice
The values for the heart to body weight ratio (mg/g)
for VDR KO and WT determined at the age of 12
months were mean 6.9 ± 1.03 and 4.9 ± 0.64, respectively
(P < 0.05).

Fig. 1. Histological analysis of heart tissues from VDR KO and WT mice. Cross-sectional and longitudinal analysis of heart myofibrils showed highly significant
cellular hypertrophy as evident by increased cell size in VDR KO mice (H&E staining). Trichrome staining of LV sections from WT and VDR KO showed
high intensity of blue staining in the heart tissue in VDR KO mice shows fibrosis which was absent in WT mice (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of the article.).
418 A. Rahman et al. / Journal of Steroid Biochemistry & Molecular Biology 103 (2007) 416–419

3.2. VDR KO mice develop cardiac hypertrophy and

fibrosis

Fig. 1 shows heart tissue using H&E staining which

revealed increased cell size. Trichrome staining showed (blue
stain) increased collagen deposition mostly in ECM suggest-
ing cardiac fibrosis in VDR KO mice.

3.3. Effect of VDR on mRNA expression of ECM

regulatory systems

In the present study, using gene microarray analysis we

observed that there were low levels of TIMP-1 and TIMP-3
gene expression in VDR KO mice as compared to WT mice.
MMP-2 and MMP-9 mRNA was also decreased as assessed
by microarray.
Fig. 2. Quantitative RT-PCR vs. microarray. Comparison of results obtained
with microarray and RT-PCR analysis of ECM gene expression profile.
Results are shown for MMP-2, MMP-9, TIMP-1, TIMP-3 for which RT-PCR
validation was performed.

3.4. ECM gene expression by quantitative real time

RT-PCR 4. Discussion

RT-PCR analysis in Fig. 2 shows an upregulation of MMP-
2 and MMP-9 gene expression with concomitant decrease
in TIMP-1 and TIMP-3 expression in VDR KO mice when
compared to WT mice, respectively.
3.5. Enhanced MMP-2 and MMP-9 activities in VDR
KO mice
Because of the discrepancy between array data and RT-
PCR we analyzed MMP-2 and MMP-9 activity. Gelatin
zymography results showed that left ventricular homogenate
from WT mice lacked MMP-2 and MMP-9 activity. However,
in left ventricular tissue from VDR KO mice, we observed
significantly high activities of MMP-2 and MMP-9 suggest-
ing the pivotal role of VDR in regulating gelatinase activities
in cardiac hypertrophy (Fig. 3).
Using the VDR KO mouse, we showed that the 1,25D
pathway plays an important role in suppressing MMP
expression in cardiac tissue. MMPs are a family of zinc
containing metalloproteinases that play an important role
in ECM degradation, synthesis and remodeling. Previous
studies have shown an increase in MMP-2 and MMP-9
gelatinolytic activity, extensive collagen deposition, and
denaturation and ECM remodeling in mice with heart
failure [11]. Several studies suggest that a positive corre-
lation exists between MMP activation and the ventricular
remodeling process and that MMP inhibition attenuates
both fibrosis and cardiac hypertrophy [8,12,13]. Previous
studies demonstrated increased expression and activity
of MMPs in animal models with systolic heart failure
[14]. The current study demonstrated that activation of
MMP-2 and MMP-9 in association with collagen deposition,

Fig. 3. Enhanced activity of MMP-2 and MMP-9 in VDR KO mice. MMP-2 and MMP-9 activities in the tissue homogenate were measured by gelatin
zymography. MMP activities were quantified using Quantity One Image Analyser (Bio-Rad) and the densitometric analysis was performed using UN-SCAN-IT
software. The graphs represent mean ± S.E.M. of three mice from each group.
 A. Rahman et al. / Journal of Steroid Biochemistry & Molecular Biology 103 (2007) 416–419
and fibrosis are associated with cellular hypertrophy in
VDR KO mice.
Increased collagen deposition was observed in VDR KO
mice with concomitant increase in MMP-2 and MMP-9
activities. It might be suggested that collagen deposition
as observed by trichrome staining may be an outcome of
MMP activation and an imbalance between ECM produc-
tion and degradation. This observation in VDR KO mice
correlates to the decreased expression of TIMPs that might
be responsible for the increased activation of MMPs. Our
study lies in concert with other studies and further extends
the observation that VDR plays important role in regulating
myocardial collagen turnover. Our previous study show that
rats fed vitamin D deficient diet have increased amounts of
collagen and collagen deposition in the extracellular space
of the myocardium [15]. Previous studies showed that cal-
citriol (activated hormonal vitamin D) modulates tissue MMP
expression [16]. Furthermore, vitamin D receptors (VDR) are
expressed in vascular wall and arterial plaque macrophages.
The MMP/TIMP system could therefore be regulated by acti-
vated hormonal vitamin D produced within vascular tissues
as well as by therapeutic agents [17]. Previous studies provide
evidence of vitamin D deficiency as a risk factor for heart dis-
eases where increased expression of circulating MMP-9 con-
tributes to pathogenesis [16]. In summary, the results of the
present study provide evidence for the role of VDR in heart
tissue remodeling in pathological situations and suggests that
VDR may be an important regulatory receptor for MMP gene
expression.
Acknowledgement
This work was supported by the National Institutes of
Health Grant #5 RO1-HL074894-02.
References
[1] G. Jones, S.A. Strugnell, H.F. DeLuca, Current understanding of
the molecular actions of vitamin D, Physiol. Rev. 78 (1998) 1193–
1231.
[2] R. Bouillon, W.H. Okamura, A.W. Norman, Structure–function rela-
tionships in the vitamin D endocrine system, Endocrinol. Rev. 16 (1995)
200–257.
419
[3] R.G. Erben, D.W. Soergiarto, K. Weber, U. Zeitz, M. Lieberherr, R.
Gniadecki, G. Moller, J. Adamski, R. Balling, Deletion of deoxyri-
bonucleic acid binding domain of the vitamin D receptor abrogates
genomic and non genomic functions of vitamin D, Mol. Endocrinol. 16
(7) (2002) 1524–1537.
[4] L.P. Zanello, A.W. Norman, Rapid modulation of osteoblast ion chan-
nel responses by 1␣,25(OH)2 D3 requires the presence of a functional
vitamin D nuclear receptor, Proc. Natl. Acad. Sci. U.S.A. 101 (6) (2004)
1589–1594.
[5] R.U. Simpson, Evidence for a specific 1␣,25-dihydroxyvitamin D3
receptor in rat heart (Abstract), Circulation 68 (1983) 239.
[6] M.R. Walters, D.C. Wickers, P.C. Riggue, 1␣,25-Dihydroxyvitamin D3
receptors identified in rat heart, J. Mol. Cell. Cardiol. 18 (1986) 67–72.
[7] R.E. Weishaar, R.U. Simpson, The involvement of endocrine sys-
tem in regulating cardiovascular function: emphasis on vitamin D3 ,
Endocrinol. Rev. 10 (1989) 351–365.
[8] F.G. Spinale, Matrix metalloproteinases: regulation and dysregulation
in the failing heart, Circ. Res. 90 (2002) 520–530.
[9] Y.Y. Li, A.M. Feldman, Y. Sun, C.F. McTiernan, Differential expression
of tissue inhibitors of metalloproteinases in the failing human heart,
Circulation 98 (1998) 1728–1734.
[10] Z. Gunja-Smith, A.R. Morales, R. Romanelli, J.F. Woessner Jr.,
Remodeling of human myocardial collagen in idiopathic dilated car-
diomyopathy. Role of metalloproteinases and pyridinoline cross-links,
Am. J. Pathol. 148 (1996) 1639–1648.
[11] Y.Y. Li, T. Kadokami, P. Wang, C.F. McTiernan, A.M. Feldman, MMP
inhibition modulates TNF-␣ transgenic mouse phenotype early in the
development of heart failure, Am. J. Physiol. Heart. Circ. Physiol. 282
(2002) H983–H989.
[12] Y.Y. Li, C.F. McTiernan, A.M. Feldman, Interplay of matrix metallo-
proteinases, tissue inhibitors of metalloproteinases and their regulators
in cardiac matrix remodeling, Cardiovasc. Res. 46 (2000) 214–224.
[13] Y.Y. Li, Y.Q. Feng, T. Kadokami, C.F. McTiernan, R. Draviam, S.C.
Watkins, A.M. Feldman, Myocardial extracellular matrix remodeling in
transgenic mice overexpressing tumor necrosis factor alpha can be mod-
ulated by anti-tumor necrosis factor alpha therapy, Proc. Natl. Acad.
Sci. U.S.A. 97 (2000) 12746–12751.
[14] V.S. Mujumdar, S.C. Tyagi, Temporal regulation of extracellular matrix
components in transition from compensatory hypertrophy to decom-
pensatory heart failure, J. Hypertens. 17 (2) (1999) 261–270.
[15] R.E. Weishaar, S.N. Kim, D. Saunders, R.U. Simpson, Involvement of
vitamin D3 with cardiovascular function. III. Effects on physical and
morphological properties, Am. J. Physiol. 258 (1990) E134–E142.
[16] D.D. Dean, Z. Schwartz, J. Schmitz, O.E. Muniz, Y. Lu, F. Calderon,
D.S. Howell, B.D. Boyan, Vitamin D regulation of metalloproteinase
activity in matrix vesicles, Connect. Tissue. Res. 35 (1–4) (1996)
331–336.
[17] P.M. Timms, N. Mannan, G.A. Hitman, K. Noonan, P.G. Mills, D.
Syndercombe-Court, E. Aganna, C.P. Price, B.J. Boucher, Circulating
MMP9, vitamin D and variation in the TIMP-1 response with VDR
genotype: mechanisms for inflammatory damage in chronic disorders?
QJM 95 (12) (2002) 787–796.