**3. Mechanism for COX2-dependent inhibition of StAR gene expression and testosterone biosynthesis in Leydig cell aging**

The studies described above indicated an involvement of COX2 in the age-related declines in StAR gene expression and testosterone biosynthesis. However, the mechanism for the COX2-dependent inhibition of StAR gene expression in Leydig cells needs to be elucidated. While further investigations are needed for the elucidation, the studies in the recent years have significantly improved the understanding of the mechanism.

#### **3.1 AA distribution between COX2 and lipoxygenase metabolic pathways**

It is known that the LH-released AA is converted to positive signals through lipoxygenase and epoxygenase pathways (Wang et al., 2006; Wang et al., 2003a), and also negative signals through COX2 pathway (Wang et al., 2003b). This suggests that StAR gene expression and testosterone biosynthesis in Leydig cell aging are regulated by the activities of these AA

Natural Flavonoids in StAR Gene Expression

**expression** 

repressors in Leydig cells remains to be clarified.

by regulating DAX-1 expression (Wang et al., 2008).

from 86 to 146 pg/µg cellular protein (Pandey et al., 2009).

and Testosterone Biosynthesis in Leydig Cell Aging 187

2002). However, whether PGF2α increases the expressions of these transcriptional

Following the studies on PGF2α, TBXAS activity was inhibited to examine the role of the COX2-TBXAS-TBX A2-receptor signaling pathway in Leydig cell steroidogenesis. Inhibition of TBXAS activity with the inhibitor furegrelate significantly enhanced the cAMP-induced steroidogenesis in MA-10 mouse Leydig cells. However, co-incubation of the cells with 22(R)hydroxycholesterol abolished the stimulatory effect of the inhibitor on steroidogenesis, suggesting that TBXAS produce an inhibitory effect on the mitochondrial cholesterol transfer. The results were confirmed by the increase in StAR protein in the cells treated with the TBXAS inhibitor (Wang et al., 2008). Following these observations, luciferase assays of StAR promoter activity and reverse transcription-polymerase chain reaction (RT-PCR) were performed to determine whether the TBXAS inhibitor acted on StAR gene transcription. It was found that inhibition of TBXAS activity significantly increased StAR promoter activity and StAR mRNA levels. To confirm the inhibitory effect of TBXAS on StAR gene expression, the TBXAS gene was silenced using RNA interference (RNAi). As TBXAS gene was silenced by RNAi, StAR mRNA, StAR protein and steroid hormone production in the cells were significantly increased. It is clear that the signaling through COX2 and TBXAS acts on StAR promoter and depresses StAR gene transcription. The study was continued to identify the transcription factor(s) that is affected by the activity of TBXAS. The results from the study reveal that inhibition of TBXAS activity reduced DAX-1 protein. When DAX-1 protein was reduced by inhibiting TBXAS activity, StAR gene transcription was significantly enhanced, suggesting that co-action of COX2 and TBXAS convert AA to inhibitory metabolite(s) that inhibits StAR gene transcription

The first AA metabolite in the COX2-TBXAS pathway is TBX A2. The TBX A2 receptors were detected in several mouse Leydig cell lines and identified on the cell surface (Pandey et al., 2009). The specific binding of the receptor antagonist SQ29548 to the receptors on MA-10 mouse Leydig cells was demonstrated by binding assay and the binding competition between 3H-SQ29548 and another receptor antagonist BM567. When the concentrations of BM567 increased from 0 to 10 µM, the 3H-SQ29548 bound to the cells was reduced to 3%. The receptor antagonist SQ29548 was used to block the binding of TBX A2 to the receptors and to determine its effect on the Leydig cell steroidogenesis. It was observed that blocking the TBX A2 receptors on the Leydig cells incubated with 0.1 mM cAMP dramatically increased StAR protein in a concentration-dependent manner. The increase in StAR protein was paralleled with the increase in steroid hormone production, with progesterone production being increased from 35 to 208 pg/µg cellular protein as the concentrations of SQ29548 were increased from 0 to 25 μM. The results were verified using another receptor antagonist BM567 (Pandey et al., 2009). Since the COX2-dependent signaling is involved in the age-related declines in StAR gene expression and testosterone biosynthesis (Wang et al., 2005), aged Leydig cells were used to determine whether blocking the TBX A2 receptors is able to reversed the declines. When the TBX A2 receptors were blocked with SQ29548, StAR protein in the aged Leydig cells increased significantly. In concomitant with the increase in StAR protein, testosterone production by the aged Leydig cells also significantly increased

**3.3 The signaling through COX2-TBXAS-TBX A2-receptor pathway in StAR gene** 

metabolic enzymes, because they control the ratio of the positive and negative signals. To prove this hypothesis, MA-10 mouse Leydig cells pre-loaded with 3H-AA were incubated with NS398 to inhibit their COX2 activities. AA metabolites were extracted from the cells and analyzed by HPLC. The HPLC profile of AA metabolites indicated that inhibition of COX2 activity increased AA metabolites produced through lipoxygenase pathway, with the levels of 5-HETE and 5-HPETE being significantly increased by the treatment. Incubation of rat Leydig cells with 5-HETE significantly increased testosterone production (Wang et al., 2005). It was previously reported that 5-HETE and 5-HPETE transduce positive signals to enhance StAR gene expression and steroidogenesis in Leydig cells (Wang et al., 2003a). The observations indicated that the distribution of AA between these two metabolic pathways is regulated by their enzyme activities. Thus, inhibition of COX2 activity changed the AA distribution, resulting in the increases in 5-HETE and 5-HPETE that enhanced StAR gene expression. It is possible that as COX2 expression increases in aged Leydig cells, more AA is metabolized through COX2 pathway to produce inhibitory metabolites that depress StAR gene expression and testosterone biosynthesis.

#### **3.2 The signaling through COX2-prostaglandin F2α-receptor pathway in StAR gene expression**

In the COX2 metabolic pathway, COX2 catalyzes AA to prostsglandin(PG)H2 that is further metabolized to PGD2, PGE2, PGF2α, PGI2 and thromboxane A2 (TBX A2) by different prostaglandin synthases and thromboxane A synthase (TBXAS). Among these metabolites, PGF2α is involved in the COX2-dependent inhibition of testosterone biosynthesis. Previous studies reported the hCG-induced production of PGF2α (Haour et al., 1979), which acted as a negative signal to inhibit testosterone production in Leydig cells (Bartke et al., 1973; Saksena et al., 1973). The inhibitory effect of PGF2α was verified by several studies. In an *in vivo* study, the infusion of PGF2α to rats (250μg/rat) for 4 hours reduced blood testosterone to 50%, whereas the inhibition of PGF2α production with indomethacin increased blood testosterone concentrations. When the decapsulated testis preparation was incubated with PGF2α, LH-induced testosterone production was reduced in a dose-dependent manner (Fuchs & Chantharaksri, 1981). The observations were corroborated by the additional studies on the inhibitory effects of PGF2α on Leydig cell steroidogenesis (Romanelli et al., 1995; Sawada et al., 1994). To find how PGF2α inhibits testosterone biosynthesis, the PGF2α receptors were located on hamster Leydig cells. Further investigation observed that incubation of the Leydig cells with 1 μM of PGF2α for 10 to 60 minutes significantly reduced StAR protein. The study described a COX-2-depenednt signaling pathway via COX2, PGF2α production, PGF2α receptors, and StAR protein in the regulation of hCG-induced testosterone biosynthesis (Frungieri et al., 2006). How this signaling pathway regulates StAR protein expression is not clear. To understand the mechanism, several regions in rat StAR promoter DNA sequences were detected to be responsive to PGF2α, including the bind sites of DAX-1, c-Fos and YY1. DAX-1 is described above as a transcriptional repressor. Injection of PGF2α for 2 hours resulted in the increase in ovarian DAX-1 protein. The increase in DAX-1 protein was associated with the 50% reduction in StAR mRNA (Sandhoff & McLean, 1999). The c-Fos binding sites were located at -85, -187 and -1561 of StAR promoter sequences. Administration of PGF2α increased c-Fos expression and reduced StAR gene transcription (Shea-Eaton et al., 2002). The YY1 binding sites were found in StAR promoter sequences at -1230/-1238, -1550/-1559 and -1651/-1660. PGF2α enhanced ovarian YY1 expression, resulting in a decrease in StAR gene expression (Liu et al., 2007; Nackley et al.,

metabolic enzymes, because they control the ratio of the positive and negative signals. To prove this hypothesis, MA-10 mouse Leydig cells pre-loaded with 3H-AA were incubated with NS398 to inhibit their COX2 activities. AA metabolites were extracted from the cells and analyzed by HPLC. The HPLC profile of AA metabolites indicated that inhibition of COX2 activity increased AA metabolites produced through lipoxygenase pathway, with the levels of 5-HETE and 5-HPETE being significantly increased by the treatment. Incubation of rat Leydig cells with 5-HETE significantly increased testosterone production (Wang et al., 2005). It was previously reported that 5-HETE and 5-HPETE transduce positive signals to enhance StAR gene expression and steroidogenesis in Leydig cells (Wang et al., 2003a). The observations indicated that the distribution of AA between these two metabolic pathways is regulated by their enzyme activities. Thus, inhibition of COX2 activity changed the AA distribution, resulting in the increases in 5-HETE and 5-HPETE that enhanced StAR gene expression. It is possible that as COX2 expression increases in aged Leydig cells, more AA is metabolized through COX2 pathway to produce inhibitory metabolites that depress StAR

**3.2 The signaling through COX2-prostaglandin F2α-receptor pathway in StAR gene** 

In the COX2 metabolic pathway, COX2 catalyzes AA to prostsglandin(PG)H2 that is further metabolized to PGD2, PGE2, PGF2α, PGI2 and thromboxane A2 (TBX A2) by different prostaglandin synthases and thromboxane A synthase (TBXAS). Among these metabolites, PGF2α is involved in the COX2-dependent inhibition of testosterone biosynthesis. Previous studies reported the hCG-induced production of PGF2α (Haour et al., 1979), which acted as a negative signal to inhibit testosterone production in Leydig cells (Bartke et al., 1973; Saksena et al., 1973). The inhibitory effect of PGF2α was verified by several studies. In an *in vivo* study, the infusion of PGF2α to rats (250μg/rat) for 4 hours reduced blood testosterone to 50%, whereas the inhibition of PGF2α production with indomethacin increased blood testosterone concentrations. When the decapsulated testis preparation was incubated with PGF2α, LH-induced testosterone production was reduced in a dose-dependent manner (Fuchs & Chantharaksri, 1981). The observations were corroborated by the additional studies on the inhibitory effects of PGF2α on Leydig cell steroidogenesis (Romanelli et al., 1995; Sawada et al., 1994). To find how PGF2α inhibits testosterone biosynthesis, the PGF2α receptors were located on hamster Leydig cells. Further investigation observed that incubation of the Leydig cells with 1 μM of PGF2α for 10 to 60 minutes significantly reduced StAR protein. The study described a COX-2-depenednt signaling pathway via COX2, PGF2α production, PGF2α receptors, and StAR protein in the regulation of hCG-induced testosterone biosynthesis (Frungieri et al., 2006). How this signaling pathway regulates StAR protein expression is not clear. To understand the mechanism, several regions in rat StAR promoter DNA sequences were detected to be responsive to PGF2α, including the bind sites of DAX-1, c-Fos and YY1. DAX-1 is described above as a transcriptional repressor. Injection of PGF2α for 2 hours resulted in the increase in ovarian DAX-1 protein. The increase in DAX-1 protein was associated with the 50% reduction in StAR mRNA (Sandhoff & McLean, 1999). The c-Fos binding sites were located at -85, -187 and -1561 of StAR promoter sequences. Administration of PGF2α increased c-Fos expression and reduced StAR gene transcription (Shea-Eaton et al., 2002). The YY1 binding sites were found in StAR promoter sequences at -1230/-1238, -1550/-1559 and -1651/-1660. PGF2α enhanced ovarian YY1 expression, resulting in a decrease in StAR gene expression (Liu et al., 2007; Nackley et al.,

gene expression and testosterone biosynthesis.

**expression** 

2002). However, whether PGF2α increases the expressions of these transcriptional repressors in Leydig cells remains to be clarified.

#### **3.3 The signaling through COX2-TBXAS-TBX A2-receptor pathway in StAR gene expression**

Following the studies on PGF2α, TBXAS activity was inhibited to examine the role of the COX2-TBXAS-TBX A2-receptor signaling pathway in Leydig cell steroidogenesis. Inhibition of TBXAS activity with the inhibitor furegrelate significantly enhanced the cAMP-induced steroidogenesis in MA-10 mouse Leydig cells. However, co-incubation of the cells with 22(R)hydroxycholesterol abolished the stimulatory effect of the inhibitor on steroidogenesis, suggesting that TBXAS produce an inhibitory effect on the mitochondrial cholesterol transfer. The results were confirmed by the increase in StAR protein in the cells treated with the TBXAS inhibitor (Wang et al., 2008). Following these observations, luciferase assays of StAR promoter activity and reverse transcription-polymerase chain reaction (RT-PCR) were performed to determine whether the TBXAS inhibitor acted on StAR gene transcription. It was found that inhibition of TBXAS activity significantly increased StAR promoter activity and StAR mRNA levels. To confirm the inhibitory effect of TBXAS on StAR gene expression, the TBXAS gene was silenced using RNA interference (RNAi). As TBXAS gene was silenced by RNAi, StAR mRNA, StAR protein and steroid hormone production in the cells were significantly increased. It is clear that the signaling through COX2 and TBXAS acts on StAR promoter and depresses StAR gene transcription. The study was continued to identify the transcription factor(s) that is affected by the activity of TBXAS. The results from the study reveal that inhibition of TBXAS activity reduced DAX-1 protein. When DAX-1 protein was reduced by inhibiting TBXAS activity, StAR gene transcription was significantly enhanced, suggesting that co-action of COX2 and TBXAS convert AA to inhibitory metabolite(s) that inhibits StAR gene transcription by regulating DAX-1 expression (Wang et al., 2008).

The first AA metabolite in the COX2-TBXAS pathway is TBX A2. The TBX A2 receptors were detected in several mouse Leydig cell lines and identified on the cell surface (Pandey et al., 2009). The specific binding of the receptor antagonist SQ29548 to the receptors on MA-10 mouse Leydig cells was demonstrated by binding assay and the binding competition between 3H-SQ29548 and another receptor antagonist BM567. When the concentrations of BM567 increased from 0 to 10 µM, the 3H-SQ29548 bound to the cells was reduced to 3%. The receptor antagonist SQ29548 was used to block the binding of TBX A2 to the receptors and to determine its effect on the Leydig cell steroidogenesis. It was observed that blocking the TBX A2 receptors on the Leydig cells incubated with 0.1 mM cAMP dramatically increased StAR protein in a concentration-dependent manner. The increase in StAR protein was paralleled with the increase in steroid hormone production, with progesterone production being increased from 35 to 208 pg/µg cellular protein as the concentrations of SQ29548 were increased from 0 to 25 μM. The results were verified using another receptor antagonist BM567 (Pandey et al., 2009). Since the COX2-dependent signaling is involved in the age-related declines in StAR gene expression and testosterone biosynthesis (Wang et al., 2005), aged Leydig cells were used to determine whether blocking the TBX A2 receptors is able to reversed the declines. When the TBX A2 receptors were blocked with SQ29548, StAR protein in the aged Leydig cells increased significantly. In concomitant with the increase in StAR protein, testosterone production by the aged Leydig cells also significantly increased from 86 to 146 pg/µg cellular protein (Pandey et al., 2009).

Natural Flavonoids in StAR Gene Expression

expression and steroidogenesis in Leydig cells

selectively inhibit COX2 activity (Cho et al., 2004; Dao et al., 2004).

dependent signaling.

**4.1 Chrysin** 

and Testosterone Biosynthesis in Leydig Cell Aging 189

has been identified, including chrysin, apigenin, luteolin, and quercetin (Fig. 1), to be able to enhance StAR gene expression and steroidogenesis in Leydig cells by blocking the COX2-

Fig. 1. Chemical structures of the flavonoids used in the experiments to enhance StAR gene

Chrysin is present in plants (Williams et al., 1997), honey and propolis (Gambelunghe et al., 2003; Jiang et al., 2008; Kassim et al., 2010). It blocks the COX2-dependent signaling principally by inhibition of COX2 expression. The reduction of COX2 by chrysin is due to its inhibitory effects on the activities of several transcription factors that regulate COX2 gene transcription in different tissues. Chrysin is able to inhibit the activity of NF-kB and reduce COX2 promoter activity (Ha et al., 2010; Li et al., 2010). In macrophages, chrysin suppressed lipopolysaccharide (LPS)-induced COX2 expression by inhibiting activity of nuclear factor for IL-6 (NF-IL6) (Woo et al., 2005). NF-IL6 is a member of the C/EBP family that binds to human COX2 promoter region at -124/-132 and induces COX-2 expression. Another member of C/EBP family is C/EBPβ that is activated by c-Jun N terminal kinase (JNK) (Cho et al., 2003). Chrysin inhibited JNK activation and reduced LPS-induced COX2 expression (Ha et al., 2010). In addition, the cytokines, such as IL-1β and TNF-α, are able to induce COX2 expression (Chen et al., 2001a; Ishikawa et al., 2005). It was observed that chrysin significantly reduced the LPS-released IL-1β and TNF-α (Ha et al., 2010; Romier et al., 2008). In addition to the inhibition of COX2 gene expression, chrysin and its derivatives are able to inhibit COX2 activity. A group of chrysin derivatives was synthesized by modification of its molecular structure. The modification significantly improves the activities of the derivatives in inhibiting COX2. Some of the chrysin derivatives are able to bind to COX2 molecule and

Chrysin has been described as a testosterone-increasing agent that blocks further testosterone metabolism by inhibiting aromatase activity (Dhawan et al., 2002; Kellis & Vickery, 1984). This testosterone-increasing activity of chrysin was confirmed in the recent studies with Leydig cells isolated from mouse and MA-10 mouse Leydig cell line, with the steroid hormone productions of the Leydig cells being significantly enhanced by chrysin

To understand how the receptor antagonists enhanced StAR protein expression and steroidogenesis, their effects on StAR gene transcription were investigated. Luciferase assays of StAR promoter activity indicated that blocking the TBX A2 receptors with the antagonists enhanced StAR promoter activity in a concentration-dependent manner in the MA-10 cells incubated with 0.1 mM cAMP. The increase in the promoter activity was associated with a similar increase in StAR mRNA level (Pandey et al., 2009). The observations indicated that blocking the TBX A2 receptor enhanced StAR gene transcription, similar to the observations on the cells treated with TBXAS inhibitor (Wang et al., 2008). Further study showed that blocking the TBX A2 receptors significantly reduced DAX-1 protein and increased StAR protein in MA-10 mouse Leydig cells. The results were confirmed with the Leydig cells isolated from aged rats. The aged Leydig cells expressed high levels of DAX-1 protein, but blocking the TBX A2 receptors dramatically reduced the DAX-1 protein, which was associated with the increases in StAR protein and testosterone production (Pandey et al., 2009). These observations indicated that the signaling through COX2-TBXAS-TBX A2-receptor inhibits StAR gene expression and testosterone biosynthesis by regulation of DAX-1 expression.

In addition, while sub-threshold levels of cAMP were unable to stimulate significant increases in StAR gene expression, interrupting the signaling through this pathway at any step, by inhibiting COX2 activity (Wang et al., 2005; Wang et al., 2003b) or TBXAS activity (Wang et al., 2008) or blocking the TBX A2 receptors (Pandey et al., 2009), reduced the threshold, with sub-threshold levels of cAMP being able to induce maximal levels of StAR protein and steroidogenesis. These studies indicated that the signaling through COX2- TBXAS-TBX A2-receptor pathway plays an important role in regulating sensitivity of Leydig cells to LH or cAMP stimulation. Therefore, when COX2 increases in Leydig cell aging, the increase in COX2 enhances the negative signaling through this pathway, which in turn reduces the sensitivity of Leydig cells and inhibits the LH-induced StAR gene expression and testosterone biosynthesis.
