Effects of heme oxygenase isozymes on Leydig cells steroidogenesis
Barbara Piotrkowski1,2,*, Casandra M Monzo ´n1,*, Romina M Pagotto1, Cecilia G Reche1, Marcos Besio1,
Cora B Cymeryng3and Omar P Pignataro1,4
1Laboratory of Molecular Endocrinology and Signal Transduction, Institute of Biology and Experimental Medicine-CONICET, Vuelta de Obligado 2490,
CP 1428 Buenos Aires, Argentina
2Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of Buenos Aires, Junin 956, 2nd Floor, CP 1113 Buenos Aires, Argentina
3Department of Human Biochemistry, CEFYBO-CONICET, School of Medicine, University of Buenos Aires, Paraguay 2155, 5th Floor, CP 1121 Buenos Aires,
4Department of Biological Chemistry, School of Sciences, University of Buenos Aires, Int Guiraldes 2160, CP 1428 Buenos Aires, Argentina
(Correspondence should be addressed to O P Pignataro at Laboratory of Molecular Endocrinology and Signal Transduction, Instituto de Biologı ´a y Medicina
Experimental (IBYME-CONICET); Email: firstname.lastname@example.org)
*(B Piotrkowski and C M Monzo ´n contributed equally to this work)
In the present study, we demonstrate the expression of heme
oxygenase (HO) isozymes, HO-1 and HO-2 (listed as
HMOX1 and HMOX2 in the MGI Database), in MA-10
Leydig tumor cells and its effect on steroidogenesis. The well-
known HO inducer, hemin, increased both HO-1 and HO-2
protein levels and HO-specific activity. Induction of HO by
hemin inhibited basal, hCG-, and dibutyryl cAMP
(db-cAMP)-induced steroidogenesis in a reversible way.
When we studied the effect of HO isozymes along the
steroid synthesis, we found that steroidogenic acute regulat-
ory protein levels were decreased, and the conversion of
cholesterol to pregnenolone was inhibited by hemin
treatment, with no changes in the content of cholesterol
side-chain cleavage enzyme (P450scc). hCG and db-cAMP
also stimulated the expression of HO-1 and HO-2, and HO
enzymatic activity in MA-10 cells. Basal and hCG-stimulated
testosterone synthesis was also inhibited by hemin in rat
normal Leydig cells. Taken together, these results suggest
that: i) at least one of HO products (presumably carbon
monoxide) inhibits cholesterol transport to the inner
mitochondrial membrane and Leydig cell steroidogenesis
by binding to the heme group of the cytochrome P450
enzymes, in a similar way as we described for nitric oxide,
and ii) hCG stimulation results in the induction of an
antioxidant enzymatic system (HO) acting as a cytoprotective
mechanism in Leydig cells, as already demonstrated in the
Journal of Endocrinology (2009) 203, 155–165
Steroid synthesis depends on two limiting steps: the first one is
at the level of the transport of cholesterol to the inner
mitochondrial membrane, a process dependent on the
steroidogenic acute regulatory protein (STAR; Stocco
2001); and the second key step is the conversion of cholesterol
to pregnenolone (P5) by the cholesterol side-chain cleavage
enzyme system (Stocco & Clark 1996). In testis, Leydig cells
are interstitial cells that synthesize testosterone through
cytochrome P450-dependent monooxygenases. Although
steroidogenesis in Leydig cells is primarily under LH control,
a number of paracrine/autocrine factors have been suggested
on steroid synthesis in Leydig cells had been previously
demonstrated in our laboratory (Del Punta et al. 1996).
Heme oxygenases (HO) catalyze the first and rate-limiting
step in the oxidative degradation of heme into three products:
carbon monoxide (CO); biliverdin; which is rapidly
converted into bilirubin by biliverdin reductase; and free
iron, which is sequestered into ferritin (Maines 1997). To
date, three isoforms have been identified: HO-1 (HMOX1),
HO-2 (HMOX2), and HO-3F (McCoubrey et al. 1997,
Maines 2005). While HO-1 (32 kDa) expression can be
induced by its own substrate heme, and several other stress
stimuli such as heavy metals, lipopolysaccharide (LPS),
inflammatory mediators, and oxidized low-density proteins
(Otterbein & Choi 2000), the other two isoforms, HO-2
(36 kDa) and HO-3 (33 kDa), are constitutively expressed
(McCoubrey et al. 1992, 1997). However, recent studies have
shown that corticosterone, estradiol, and a photic signal can
induce HO-2 expression in testis (Liu et al. 2000), endothelial
cells (Tschugguel et al. 2001), and retina (Sacca et al. 2003)
respectively. Both HO-1 and HO-2 isoforms have been
Journal of Endocrinology (2009) 203, 155–165
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detected in diverse organs, including reproductive ones such
as testes (Trakshel & Maines 1988, Ewing & Maines 1995),
placenta (Lyall et al. 2000), uterus (Acevedo & Ahmed 1998),
and ovary (Alexandreanu & Lawson 2003).
CO is a gaseous second messenger that shares several
biological properties with NO, including the activation of
guanylate cyclase, signal transduction, and gene regulation
(Verma et al. 1993, Zhuo et al. 1993). Based on the similarities
with NO, recent studies have suggested a possible regulatory
role for CO on steroid production in ovary and adrenal gland
(Alexandreanu & Lawson 2003, Pomeraniec et al. 2004).
HO-1 in Leydig cells modulated spermatogenesis and
triggered apoptosis of germ cells under stress conditions
(Ozawa et al. 2002). Besides, in humans, increased HO-1
expression in Leydig cells improved spermatogenesis in
varicocele condition (Shiraishi & Naito 2005), and HO-
1-derived CO in testicular Sertoli cells may have a functional
in the seminiferous tubule (Middendorff et al. 2000).
Furthermore, numerous studies proposed that the activity of
stress (Stocker 1990, Maines 1997, Niess et al. 1999,
Pomeraniec et al. 2004, Shiraishi & Naito 2005).
Although HO-1 and HO-2 isoforms have been detected
in the rat testes, the biochemical mechanisms by which
the HO/CO system regulates steroidogenesis have not
been investigated so far. So, the aim of this study was to
analyze the expression levels of both HO isoforms and
the influence of HO activityon steroid production in MA-10
Materials and Methods
Purified hCG (CR-127, 14; 900 IU/mg) was a gift from the
National Hormone and Pituitary Program, National Institute
of Diabetes and Digestive and Kidney Diseases (Bethesda,
MD, USA). The specific antibody for progesterone (P4) and
testosterone was a gift from Dr G D Niswender (Animal
Reproduction and Biotechnology Lab, Colorado State
University, Fort Collins, CO, USA). HO-1 and HO-2
antibodies were from StressGen Biotechnologies Corp.
(Victoria, BC, Canada). STAR and cytochrome P450scc
(P450scc or CYP11A) antibodieswereagift fromDr Walter L
Miller (University of California, San Francisco) and Dr Dale
B Hales (University of Illinois at Chicago) respectively.
Peroxidase-conjugated anti-IgG antibodies were purchased
from Amersham Pharmacia. Cell culture supplies and plastic
ware were obtained from Gibco-BRL and Corning
(Corning, NY, USA) respectively. Hemin (a well-known
HO inducer) and dibutyryl cAMP (db-cAMP, the permeable
analog of the second messenger) were purchased from Sigma.
Collagenase was from Worthington (Freehold, NJ, USA).
Other reagents used were of the best grade available and were
obtained from commonly used suppliers.
Cellular culture of MA-10 Leydig cells
The MA-10 cell line (kindly provided by Mario Ascoli,
University of Iowa, Ames, IA, USA) is a clonal strain of
Leydig tumor cells that secrete P4rather than testosterone as a
major steroid. This cell line provides a suitable model system
for the study of gonadotropin actions and regulation of
differentiated functions of Leydig cells, as they behave like
normal steroidogenic cells in several aspects, including the
stimulation of steroid production by LH/hCG in a cAMP-
dependent pathway. The origin and handling of MA-10 cells
have already been described (Ascoli 1981, Pignataro & Ascoli
1990b). Cells were plated in 100-mm Petri dish plates (for
immunoblot analysis and HO activity) or in 24!16-mm well
plates (for steroidogenesis experiments) on day 0 at a density
of 3!106cells/dish or 1.25!105cells/well, and in a total
volume of 10 or 1 ml of growth medium (Waymouth
MB752/1, modified to contain 1.1 g/l NaHCO3, 20 mmol/l
Hepes, 50 mg/ml gentamycin, and 15% (v/v) horse serum,
pH 7.4) respectively. The cells were maintained in a
humidified atmosphere containing 5% CO2and were used
on day 3. At this time, the cell density was w10!106cells/
dish or 5!105cells/well. On this day, the cells were washed
with 1 ml warm serum-free medium supplemented with
1 mg/ml BSA (assay medium). Incubations were performed
in a total volume of 7 ml (for dishes) or 0.5 ml (for wells) assay
medium at 37 8C with the corresponding additions as
described in each figure. After 5 h (unless other indicated),
media were collected, and P4 was measured by RIA
(Pignataro & Ascoli 1990a). The intra- and inter-assay
variations were 8.0 and 14.2% respectively. Cells were treated
as describes below. When hemin was used for experiments,
30-min pretreatment with the compound was done.
To study the reversibility of the inhibitory effect of the HO
inducer on steroid synthesis, MA-10 cells were incubated in
the absence or presence of 10 mmol/l hemin with or without
0.2 mmol/l db-cAMP. After 5 h, media were collected for P4
determination (day 1). Cells were washed and incubated with
hemin-free fresh medium for an additional 24 h. By the end
of this incubation period, cellswerestimulatedwith 1 mmol/l
db-cAMP for 5 h (day 2).
Rat Leydig cell isolation and testosterone production
For all the experiments, Leydig cells were isolated from a
pool of 16 testes obtained from eight adult Sprague–Dawley
rats (60 days old, 200–250 g, Charles River descendants,
Animal Care Lab, IByME, Buenos Aires, Argentina), as
previously described (Charreau et al. 1981, Pignataro et al.
1983, Mondillo et al. 2009). Animals were housed in groups
in an air-conditioned room with lights on from 0700 to
1900 h. They were given free access to laboratory chow and
tap water. Animals were killed by CO2asphyxia according to
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Received in final form 28 July 2009
Accepted 31 July 2009
Made available online as an Accepted Preprint
31 July 2009
Heme oxygenases isozymes and steroidogenesis .
B PIOTRKOWSKI, C M MONZO´N and others165
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