**4.1 Chrysin**

188 Basic and Clinical Endocrinology Up-to-Date

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

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

**4. Flavonoid intervention in the COX2-dependent inhibition of StAR gene** 

To study the possibility of delaying the decline in blood testosterone by intervention in the mechanism, aged rats were fed with increasing concentrations of a selective COX2 inhibitor mixed in their diet. After 30 days, StAR protein in their Leydig cells increased in a concentration-dependent manner. The blood testosterone concentrations increased up to 120% over control (Wang et al., 2005). The studies suggest a possibility of delaying the agerelated declines in StAR protein and testosterone using COX2 inhibitors. However, longterm application of the COX2 inhibitors currently used in the clinical practice is limited by their side effects. Therefore, alternative approaches are needed for the health of aging males. In the recent years, steroidogenic effects of natural flavonoids have been studied with Leydig cells. Flavonoids are a group of the polyphenolic compounds that are widely distributed in various food and food supplements, especially in fruits and vegetables. Previous studies have reported the activities of flavonoids in anti-inflammation, anti-cancer, and anti-oxidation (Cardenas et al., 2006; Chen et al., 1990; Ferrandiz & Alcaraz, 1991). One of the important mechanisms for these activities is the inhibition of COX2 expression and blocking the COX2-dependent signaling by flavonoids, which enables flavonoids to enhance StAR gene expression and testosterone biosynthesis in Leydig cells. A group of flavonoids

by regulation of DAX-1 expression.

and testosterone biosynthesis.

**expression and testosterone biosynthesis** 

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

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

Natural Flavonoids in StAR Gene Expression

Leydig cells (Li et al., 2011).

**4.3 Luteolin** 

and Testosterone Biosynthesis in Leydig Cell Aging 191

from mouse. The binding of apigenin to the TBX A2 receptors was examined. The binding competition between apigenin and the selective receptor antagonist 3H-SQ29548 indicated that this flavonoid specifically blocked the TBX A2 receptors on MA-10 mouse Leydig cells. Blocking the receptors with apigenin significantly increased StAR protein and testosterone production in mouse Leydig cells (Li et al., 2011). In MA-10 Leydig cells, apigenin induced concentration-dependent increases in StAR promoter activity, StAR mRNA, StAR protein and steroid hormone production. Further study indicated that this flavonoid enhanced StAR gene transcription by reduction of the transcriptional repressor, DAX-1 protein. While the mechanism for the reduction of DAX-1 protein in apigenin-treated cells is unknown, the results from the study suggest that minimal levels of PKA and protein kinase C (PKC) activities are essential for the effects of apigenin on DAX-1, StAR and steroidogenesis in

Luteolin is found in fruits and vegetables (Harnly et al., 2006). It was detected in bird chili (1035.0 mg/kg, in dry weight), belimbi leaves (464.5 mg/kg), onion leaves (391.0 mg/kg), belimbi fruit (202.0 mg/kg), dried asam gelugur (107.5 mg/kg), local celery (80.5 mg/kg), broccoli (74.5 mg/kg), carrot (37.5 mg/kg), limau purut leaves (30.5 mg/kg), French bean (11.0 mg/kg), and white radish (9.0 mg/kg) (Miean & Mohamed, 2001). Similar to the effects of apigenin, luteolin is able to inhibit COX2 expression and block the TBX A2 receptors. It was reported that luteolin reduced COX2 expression by inhibiting NF-kB activity (Chen et al., 2007a; Gutierrez-Venegas et al., 2006; Kim & Jobin, 2005), through the mechanism involving the luteolin-reduced IkB kinase (IKK) activity, IkB degradation, nuclear translocation of NF-kB p65 subunit, and NF-kB-binding to the promoter DNA. Luteolin may reduce COX2 expression by inhibiting activity of MAPK and subsequently reducing the activities of the transcription factors that are essential for COX2 transcription (Choi & Lee, 2010; Gutierrez-Venegas et al., 2006). It is known that COX2 expression is up-regulated by inflammatory cytokines, such as IL-1β and TNF-α. Luteolin was reported being able to reduce these cytokines, which may contribute to its inhibitory effect on COX2 expression (Wu et al., 2009). In addition, luteolin is also a natural antagonist of the TBX A2 receptors. It blocked the COX2-dependent signaling through COX2-TBXAS-TBX A2-receptor pathway

The inhibitory effect of luteolin on COX2-dependent signaling suggests a possibility of using this flavonoid to enhance StAR gene expression and steroidogenesis in Leydig cells. This possibility was examined by the experiments with MA-10 mouse Leydig cells cultured for 6 hours in the medium containing increasing concentrations of luteolin and a cAMP analog (dbcAMP). StAR and DAX-1 proteins, steroid production, StAR mRNA and StAR promoter activity were analyzed as described in the previous study (Jana et al., 2008). As shown in Fig. 2A, the treatments with luteolin induced a concentration-dependent increase in steroid hormone production. Progesterone concentrations in culture medium were increased from 4.9 to 124.7ng/ml, as the levels of luteolin in the culture were increased from 0 to 12 µM. In the presence of 22(R)hydroxycholesterol(22R), there was no significant difference in steroid production among the treatments, suggesting that luteolin increase steroidogenesis by improving mitochondrial cholesterol transfer in Leydig cells. The results were enhanced by the luteolin-increased StAR protein expression. Similar increases were observed in the analyses of StAR promoter activity and StAR mRNA levels (Fig. 2B), indicating a regulatory effect of luteolin on StAR gene transcription. In addition, a synergistic interaction between

by binding to the receptors (Guerrero et al., 2005; Guerrero et al., 2007).

(Jana et al., 2008). However, it failed to induce the significant increase in steroid hormone when MA-10 Leydig cells were co-incubated with 22(R)hydroxycholesterol, suggesting that this flavonoid increase steroidogenesis mainly by improving the cholesterol transfer to the mitochondrial inner membrane. This was proved by the results from Western blot analyses that showed a significant increase in StAR protein in the Leydig cells treated with chrysin. The observations were corroborated by the significant increases in StAR promoter activity and StAR mRNA levels in the cells, indicating that chrysin acted on StAR promoter and enhanced StAR gene transcription. The transcription factors that act on StAR promoter were examined in the Leydig cells incubated with chrysin. It was found that this flavonoid significantly reduced the transcriptional repressor, DAX-1 protein (Jana et al., 2008). As DAX-1 protein and DAX-1 mRNA were reduced, StAR protein and steroid hormone production dramatically increased. Similar to the effect of COX2 inhibitors, chrysin did not affect PKA activity. In the absence of cAMP, this flavonoid alone was not able to increase StAR protein and steroidogenesis. However, in the presence of 10 µm chrysin, sensitivity of Leydig cells dramatically increased, with sub-threshold levels of cAMP being able to induce maximal levels of StAR protein and steroidogenesis. The studies indicated that chrysin enhanced steroidogenesis in Leydig cells mainly by enhancing cAMP-induced StAR gene expression.

#### **4.2 Apigenin**

Apigenin is present in various plants at different concentrations (Miean & Mohamed, 2001). It was found in parsley at high level (Nielsen et al., 1999). This flavonoid blocks COX2 dependent signaling at two separated steps, by inhibiting COX2 expression and blocking the TBX A2 receptors. It inhibits COX2 expression at the levels of transcription and translation. At the transcriptional level, apigenin inhibits activities of the transcription factors that are important for COX2 gene expression. Previous studies reported that incubation of mouse macrophages with apigenin reduced COX2 expression by inhibition of NF-kB activation, through a mechanism involving the apigenin-reduced degradation of the inhibitor kB (IkB) (Liang et al., 1999). This observation was enhanced by the study on the apigenin-reduced COX2 expression, in which apigenin-inhibited IkBα degradation resulted in reductions in NF-kB-binding to the promoter DNA and the TNF-α-induced COX2 expression (Shukla & Gupta, 2004). In addition, apigenin is able to inhibit the activation of MAPK. It is known that MAPK activation is essential for the activities of several transcription factors, such as CREB, NF-kB, and C/EBP, that induce COX2 expression in various tissues (Chun & Surh, 2004; Tsatsanis et al., 2006). The inhibitory effects of apigenin on MAPK activation significantly reduced COX2 gene transcription (Ha et al., 2008; Yi Lau & Leung, 2010). At the translational level, apigenin increased the localization to cytoplasm of two proteins, HuR and T-cell-restricted intracellular antigen 1-related protein (TIAR). HuR and TIAR then bound to the AU-rich elements in the 3'-untranslated region of COX2 mRNA and inhibited COX2 translation (Tong et al., 2007). In addition to its inhibitory effects on COX2 expression, apigenin acts as a natural antagonist of the TBX A2 receptors. Binding of apigenin to the receptors blocked the signaling through COX2-TBXAS-TBX A2-receptors pathway (Guerrero et al., 2007; Navarro-Nunez et al., 2008).

It was reported that blocking the signaling at any step of the COX2-TBXAS-TBX A2 receptors pathway significantly increased StAR gene expression and testosterone biosynthesis in Leydig cells (Pandey et al., 2009). Therefore, the effects of apigenin on steroidogenesis were studied using MA-10 mouse Leydig cell line and Leydig cells isolated

from mouse. The binding of apigenin to the TBX A2 receptors was examined. The binding competition between apigenin and the selective receptor antagonist 3H-SQ29548 indicated that this flavonoid specifically blocked the TBX A2 receptors on MA-10 mouse Leydig cells. Blocking the receptors with apigenin significantly increased StAR protein and testosterone production in mouse Leydig cells (Li et al., 2011). In MA-10 Leydig cells, apigenin induced concentration-dependent increases in StAR promoter activity, StAR mRNA, StAR protein and steroid hormone production. Further study indicated that this flavonoid enhanced StAR gene transcription by reduction of the transcriptional repressor, DAX-1 protein. While the mechanism for the reduction of DAX-1 protein in apigenin-treated cells is unknown, the results from the study suggest that minimal levels of PKA and protein kinase C (PKC) activities are essential for the effects of apigenin on DAX-1, StAR and steroidogenesis in Leydig cells (Li et al., 2011).

#### **4.3 Luteolin**

190 Basic and Clinical Endocrinology Up-to-Date

(Jana et al., 2008). However, it failed to induce the significant increase in steroid hormone when MA-10 Leydig cells were co-incubated with 22(R)hydroxycholesterol, suggesting that this flavonoid increase steroidogenesis mainly by improving the cholesterol transfer to the mitochondrial inner membrane. This was proved by the results from Western blot analyses that showed a significant increase in StAR protein in the Leydig cells treated with chrysin. The observations were corroborated by the significant increases in StAR promoter activity and StAR mRNA levels in the cells, indicating that chrysin acted on StAR promoter and enhanced StAR gene transcription. The transcription factors that act on StAR promoter were examined in the Leydig cells incubated with chrysin. It was found that this flavonoid significantly reduced the transcriptional repressor, DAX-1 protein (Jana et al., 2008). As DAX-1 protein and DAX-1 mRNA were reduced, StAR protein and steroid hormone production dramatically increased. Similar to the effect of COX2 inhibitors, chrysin did not affect PKA activity. In the absence of cAMP, this flavonoid alone was not able to increase StAR protein and steroidogenesis. However, in the presence of 10 µm chrysin, sensitivity of Leydig cells dramatically increased, with sub-threshold levels of cAMP being able to induce maximal levels of StAR protein and steroidogenesis. The studies indicated that chrysin enhanced steroidogenesis in Leydig cells mainly by enhancing cAMP-induced StAR gene

Apigenin is present in various plants at different concentrations (Miean & Mohamed, 2001). It was found in parsley at high level (Nielsen et al., 1999). This flavonoid blocks COX2 dependent signaling at two separated steps, by inhibiting COX2 expression and blocking the TBX A2 receptors. It inhibits COX2 expression at the levels of transcription and translation. At the transcriptional level, apigenin inhibits activities of the transcription factors that are important for COX2 gene expression. Previous studies reported that incubation of mouse macrophages with apigenin reduced COX2 expression by inhibition of NF-kB activation, through a mechanism involving the apigenin-reduced degradation of the inhibitor kB (IkB) (Liang et al., 1999). This observation was enhanced by the study on the apigenin-reduced COX2 expression, in which apigenin-inhibited IkBα degradation resulted in reductions in NF-kB-binding to the promoter DNA and the TNF-α-induced COX2 expression (Shukla & Gupta, 2004). In addition, apigenin is able to inhibit the activation of MAPK. It is known that MAPK activation is essential for the activities of several transcription factors, such as CREB, NF-kB, and C/EBP, that induce COX2 expression in various tissues (Chun & Surh, 2004; Tsatsanis et al., 2006). The inhibitory effects of apigenin on MAPK activation significantly reduced COX2 gene transcription (Ha et al., 2008; Yi Lau & Leung, 2010). At the translational level, apigenin increased the localization to cytoplasm of two proteins, HuR and T-cell-restricted intracellular antigen 1-related protein (TIAR). HuR and TIAR then bound to the AU-rich elements in the 3'-untranslated region of COX2 mRNA and inhibited COX2 translation (Tong et al., 2007). In addition to its inhibitory effects on COX2 expression, apigenin acts as a natural antagonist of the TBX A2 receptors. Binding of apigenin to the receptors blocked the signaling through COX2-TBXAS-TBX A2-receptors pathway

It was reported that blocking the signaling at any step of the COX2-TBXAS-TBX A2 receptors pathway significantly increased StAR gene expression and testosterone biosynthesis in Leydig cells (Pandey et al., 2009). Therefore, the effects of apigenin on steroidogenesis were studied using MA-10 mouse Leydig cell line and Leydig cells isolated

expression.

**4.2 Apigenin** 

(Guerrero et al., 2007; Navarro-Nunez et al., 2008).

Luteolin is found in fruits and vegetables (Harnly et al., 2006). It was detected in bird chili (1035.0 mg/kg, in dry weight), belimbi leaves (464.5 mg/kg), onion leaves (391.0 mg/kg), belimbi fruit (202.0 mg/kg), dried asam gelugur (107.5 mg/kg), local celery (80.5 mg/kg), broccoli (74.5 mg/kg), carrot (37.5 mg/kg), limau purut leaves (30.5 mg/kg), French bean (11.0 mg/kg), and white radish (9.0 mg/kg) (Miean & Mohamed, 2001). Similar to the effects of apigenin, luteolin is able to inhibit COX2 expression and block the TBX A2 receptors. It was reported that luteolin reduced COX2 expression by inhibiting NF-kB activity (Chen et al., 2007a; Gutierrez-Venegas et al., 2006; Kim & Jobin, 2005), through the mechanism involving the luteolin-reduced IkB kinase (IKK) activity, IkB degradation, nuclear translocation of NF-kB p65 subunit, and NF-kB-binding to the promoter DNA. Luteolin may reduce COX2 expression by inhibiting activity of MAPK and subsequently reducing the activities of the transcription factors that are essential for COX2 transcription (Choi & Lee, 2010; Gutierrez-Venegas et al., 2006). It is known that COX2 expression is up-regulated by inflammatory cytokines, such as IL-1β and TNF-α. Luteolin was reported being able to reduce these cytokines, which may contribute to its inhibitory effect on COX2 expression (Wu et al., 2009). In addition, luteolin is also a natural antagonist of the TBX A2 receptors. It blocked the COX2-dependent signaling through COX2-TBXAS-TBX A2-receptor pathway by binding to the receptors (Guerrero et al., 2005; Guerrero et al., 2007).

The inhibitory effect of luteolin on COX2-dependent signaling suggests a possibility of using this flavonoid to enhance StAR gene expression and steroidogenesis in Leydig cells. This possibility was examined by the experiments with MA-10 mouse Leydig cells cultured for 6 hours in the medium containing increasing concentrations of luteolin and a cAMP analog (dbcAMP). StAR and DAX-1 proteins, steroid production, StAR mRNA and StAR promoter activity were analyzed as described in the previous study (Jana et al., 2008). As shown in Fig. 2A, the treatments with luteolin induced a concentration-dependent increase in steroid hormone production. Progesterone concentrations in culture medium were increased from 4.9 to 124.7ng/ml, as the levels of luteolin in the culture were increased from 0 to 12 µM. In the presence of 22(R)hydroxycholesterol(22R), there was no significant difference in steroid production among the treatments, suggesting that luteolin increase steroidogenesis by improving mitochondrial cholesterol transfer in Leydig cells. The results were enhanced by the luteolin-increased StAR protein expression. Similar increases were observed in the analyses of StAR promoter activity and StAR mRNA levels (Fig. 2B), indicating a regulatory effect of luteolin on StAR gene transcription. In addition, a synergistic interaction between

Natural Flavonoids in StAR Gene Expression

by reduction of DAX-1 expression.

DAX-1 protein

formation (Garcia-Saura et al., 2005; Sheu et al., 2004).

and Testosterone Biosynthesis in Leydig Cell Aging 193

signaling. While LPS activates the MAPKs, quercetin inhibited the activation of each of them, and subsequently reduced IκB phosphorylation and COX2 expression (Gutierrez-Venegas et al., 2007). The observations were enhanced by the study on the inhibitory effects of quercetin on ROS-induced MAPK activation and COX2 expression (Huang et al., 2006). The inhibition of MAPKs by quercetin was associated with the reductions in inflammatory cytokines, such as IL-1β and TNF-α (Overman et al., 2011). In addition, quercetin is able to block the COX2-TBXAS-TBX A2-receptor signaling pathway by inhibition of TBX A2

The steroidogenic effect of quercetin was studied with MA-10 mouse Leydig cells. It was reported that quercetin increased StAR mRNA levels, StAR promoter activity and steroid hormone production (Chen et al., 2007c). The observations were corroborated by the results shown in Fig. 3., which indicated the quercetin-enhanced StAR gene transcription and translation in MA-10 cells cultured for 6 hours (Fig. 3 A and B). Following these studies, the transcription factors in StAR gene transcription were examined. It was found that the incubation of MA-10 Leydig cells with quercetin reduced DAX-1 protein, similar to the effects of other flavonoids. The reduction in DAX-1 protein was associated with a dramatic increase in StAR protein (Fig. 3C), suggesting that quercetin enhance StAR gene expression

Fig. 3. Effect of quercetin on StAR gene expression and steroidogenesis in MA-10 mouse Leydig cells. MA-10 cells were incubated with quercetin for 6 hours, and then collected for analyses: A, StAR protein and steroidogenesis; B, StAR gene transcription; C, Reduction in

luteolin and cAMP was observed (Fig. 2C), in which luteolin reduced the threshold of cAMP-induced StAR gene expression and increased the sensitivity of Leydig cells to cAMP stimulation. Further study indicated that luteolin enhanced StAR gene transcription by inhibiting DAX-1 expression (Fig. 2D), similar to the observation with apigenin.

Fig. 2. Effect of luteolin on StAR gene expression and steroidogenesis in MA-10 mouse Leydig cells. MA-10 cells were incubated with luteolin for 6 hours, and then collected for analyses: A, StAR protein and steroidogenesis; B, StAR gene transcription; C, Interaction between luteolin and cAMP; D, Reduction in DAX-1 protein.

#### **4.4 Quercetin**

Quercetin was reported as one of the major flavonoids in the plants (Miean & Mohamed, 2001), especially in onions (Slimestad et al., 2007). It blocks the COX2-dependent signaling by inhibiting COX2 expression and TBX A2 production. Similar to other flavonoids, quercetin depresses COX2 expression by inhibiting activities of NF-kB. It was observed that this flavonoid reduced the activity of NF-kB by inhibiting IKK/IκB signaling cascade (Garcia-Mediavilla et al., 2007), which in turn reduced NF-kB nuclear translocation, its binding to promoter, and COX2 expression (Crespo et al., 2008; Puangpraphant & de Mejia, 2009). Another signaling pathway regulating COX2 expression is PI3K-Akt pathway. This signaling pathway may activate CREB (Alique et al., 2011) or NF-kB (Yang et al., 2009), and induce COX2 expression. A recent study found that quercetin directly bound with PI3K to inhibit PI3K activity and Akt phosphorylation, resulting in a reduction in COX2 expression (Lee et al., 2010). In the LPS-induced COX2 expression, the members of MAPK, including extracellular signal related kinase (ERK), p38 and JNK, are involved in the LPS-induced

luteolin and cAMP was observed (Fig. 2C), in which luteolin reduced the threshold of cAMP-induced StAR gene expression and increased the sensitivity of Leydig cells to cAMP stimulation. Further study indicated that luteolin enhanced StAR gene transcription by

inhibiting DAX-1 expression (Fig. 2D), similar to the observation with apigenin.

Fig. 2. Effect of luteolin on StAR gene expression and steroidogenesis in MA-10 mouse Leydig cells. MA-10 cells were incubated with luteolin for 6 hours, and then collected for analyses: A, StAR protein and steroidogenesis; B, StAR gene transcription; C, Interaction

Quercetin was reported as one of the major flavonoids in the plants (Miean & Mohamed, 2001), especially in onions (Slimestad et al., 2007). It blocks the COX2-dependent signaling by inhibiting COX2 expression and TBX A2 production. Similar to other flavonoids, quercetin depresses COX2 expression by inhibiting activities of NF-kB. It was observed that this flavonoid reduced the activity of NF-kB by inhibiting IKK/IκB signaling cascade (Garcia-Mediavilla et al., 2007), which in turn reduced NF-kB nuclear translocation, its binding to promoter, and COX2 expression (Crespo et al., 2008; Puangpraphant & de Mejia, 2009). Another signaling pathway regulating COX2 expression is PI3K-Akt pathway. This signaling pathway may activate CREB (Alique et al., 2011) or NF-kB (Yang et al., 2009), and induce COX2 expression. A recent study found that quercetin directly bound with PI3K to inhibit PI3K activity and Akt phosphorylation, resulting in a reduction in COX2 expression (Lee et al., 2010). In the LPS-induced COX2 expression, the members of MAPK, including extracellular signal related kinase (ERK), p38 and JNK, are involved in the LPS-induced

between luteolin and cAMP; D, Reduction in DAX-1 protein.

**4.4 Quercetin** 

signaling. While LPS activates the MAPKs, quercetin inhibited the activation of each of them, and subsequently reduced IκB phosphorylation and COX2 expression (Gutierrez-Venegas et al., 2007). The observations were enhanced by the study on the inhibitory effects of quercetin on ROS-induced MAPK activation and COX2 expression (Huang et al., 2006). The inhibition of MAPKs by quercetin was associated with the reductions in inflammatory cytokines, such as IL-1β and TNF-α (Overman et al., 2011). In addition, quercetin is able to block the COX2-TBXAS-TBX A2-receptor signaling pathway by inhibition of TBX A2 formation (Garcia-Saura et al., 2005; Sheu et al., 2004).

The steroidogenic effect of quercetin was studied with MA-10 mouse Leydig cells. It was reported that quercetin increased StAR mRNA levels, StAR promoter activity and steroid hormone production (Chen et al., 2007c). The observations were corroborated by the results shown in Fig. 3., which indicated the quercetin-enhanced StAR gene transcription and translation in MA-10 cells cultured for 6 hours (Fig. 3 A and B). Following these studies, the transcription factors in StAR gene transcription were examined. It was found that the incubation of MA-10 Leydig cells with quercetin reduced DAX-1 protein, similar to the effects of other flavonoids. The reduction in DAX-1 protein was associated with a dramatic increase in StAR protein (Fig. 3C), suggesting that quercetin enhance StAR gene expression by reduction of DAX-1 expression.

Fig. 3. Effect of quercetin on StAR gene expression and steroidogenesis in MA-10 mouse Leydig cells. MA-10 cells were incubated with quercetin for 6 hours, and then collected for analyses: A, StAR protein and steroidogenesis; B, StAR gene transcription; C, Reduction in DAX-1 protein

Natural Flavonoids in StAR Gene Expression

testosterone biosynthesis.

**6. Acknowledgment** 

**7. References** 

expression and testosterone biosynthesis.

No.2, pp. 147-153.

mesangial cells. *Am J Physiol Cell Physiol*.

**5. Summary** 

and Testosterone Biosynthesis in Leydig Cell Aging 195

(Setchell et al., 2001; Setchell et al., 2002). Specifically in the flavonoid-enhanced StAR gene expression and testosterone production, the following factors have been discussed recently: 1) Biphasic effects of flavonoids on StAR gene expression. As mentioned above, some of the flavonoids are able to block L-type calcium channels. It was reported that blocking L-type calcium channel produced biphasic effects on StAR gene expression in Leydig cells (Pandey et al., 2010). While these flavonoids might enhance StAR gene expression by reducing the transcriptional repressor DAX-1 protein, they might inhibit the positive effect of calciumdependent signaling on StAR gene expression; 2) Inhibitory effects of flavonoids on steroidogenic enzymes. It was reported that some of the flavonoids are able to inhibit activities of the enzymes involved in testosterone biosynthesis (Figueiroa et al., 2009; Hu et al., 2010). These inhibitory effects may reduce the efficacy of flavonoid-enhanced testosterone production; 3) Levels of cAMP in Leydig cells. The levels of cAMP-PKAphosphorylation are critical for the flavonoid-enhanced StAR gene expression in Leydig cells. As shown in Fig. 2C, in the absence of cAMP, flavonoid alone is not able to induce significant increases in StAR protein expression and steroid hormone production (Jana et al., 2008; Li et al., 2011). In the studies without exogenous cAMP, the endogenous cAMP becomes important for the steroidogenic effect of flavonoids. When the levels of endogenous cAMP or PKA activity in Leydig cells are not sufficient, flavonoid might not be able to induce significant increase in StAR gene expression. Therefore, minimal level of cAMP or PKA activity should be considered for the flavonoid-enhanced StAR gene expression and

In summary, LH-stimulation of Leydig cells induces both positive and negative signals in the regulation of StAR gene expression and testosterone biosynthesis. In the aging process, the increase in COX2 enhances the negative signaling, resulting in the declines in StAR gene expression and testosterone biosynthesis in Leydig cells. It was found that some of the flavonoids are able to block the COX2-dependent signaling and enhance StAR gene expression. While further investigations are needed, the studies suggest a potential for application of the natural flavonoids in delaying the age-related declines in StAR gene

Abayasekara, D.R., Band, A.M., & Cooke, B.A. (1990). Evidence for the involvement of

Alique, M., Calleros, L., Luengo, A., Griera, M., Iniguez, M.A., Punzon, C., Fresno, M.,

phospholipase A2 in the regulation of luteinizing hormone-stimulated steroidogenesis in rat testis Leydig cells. *Molecular Cellular Endocrinology,* Vol.70,

Rodriguez-Puyol, M., & Rodriguez-Puyol, D. (2011). Changes in extracellular matrix composition regulate cyclooxygenase- 2 (COX-2) expression in human

The author would like to acknowledge the support of NIH Grant AG025349.

#### **4.5 Potential application of natural flavonoids in delaying the declines in StAR gene expression and testosterone biosynthesis**

The studies reviewed above suggest that flavonoids are potential resources for alternative medicine to delay the decline in StAR gene expression. Among the various compounds of natural flavonoids, many of them are able to reduce COX2 expression, such as Kaempferol (Garcia-Mediavilla et al., 2007), tectorigenin, tectoridin (Kim et al., 1999), wogonin (Chen et al., 2008) and Silibinin (Kim et al., 2009). Some of the flavonoids reduce the inflammatory factors that induce COX2 expression (Garcia-Lafuente et al., 2009; Khanna et al., 2007; Kim et al., 2004). Another group of flavonoids was reported as receptor antagonists that inhibit the COX2-dependent signaling by blocking the TBX A2-binding to the receptors (Navarro-Nunez et al., 2009). In addition to the COX2-dependent signaling, StAR gene expression is affected by the signaling through different pathways. For example, calcium influx through L-type calcium channels regulates the threshold of cAMP-induced StAR gene expression, so that blocking the L-type calcium channels reduced the threshold and enhanced StAR gene expression in Leydig cells (Pandey et al., 2010). There is a group of natural flavonoids that are able to block the L-type calcium channels, such as genistein (Belevych et al., 2002), daidzein (Yokoshiki et al., 1996), equol (Liew et al., 2003) and epigallocatechin-3-gallate (EGCG) (Kang et al., 2010). The steroidogenic effects of the flavonoids in this group were described (Yu et al., 2010). It is possible to identify different flavonoids that might enhance StAR gene expression and testosterone biosynthesis through different mechanisms.

The potential for application of natural flavonoids in delaying the decline in testosterone is supported by the animal studies on the flavonoid-enhanced testosterone production and reproductive function. A study with 2-year-old male rats reported that supplementation with chrysin improved their reproductive functions, with their sperm count, fertilization potential and litter size being increased when they were allowed to interact with female rats (Dhawan et al., 2002). It was also observed that lifetime exposure to the flavonoids, including daidzein, glycitein and genistein, increased serum and testicular testosterone concentrations of rats (McVey et al., 2004). The observation was enhanced by the study on the effects of catechins on testosterone biosynthesis in rats, in which injection with catechins or its derivatives increased testosterone in blood (Yu et al., 2010). A recent study reported the beneficial effects of quercetin in diabetic rats, with their sperm viability and motility being improved by the flavonoid. These effects of quercetin are associated with an increase in serum total testosterone (Khaki et al., 2010). In addition, some flavonoids in herbal medicine are able to enhance testosterone production in rats. For example, icariin, a flavonoid in the plants in *Epimedium* family, has been extracted from traditional Chinese medicine (Du et al., 2002). It was observed that supplementation of icariin to 15-month-old male rats significantly increased their blood testosterone levels (Zhang & Yang, 2006).

For application of natural flavonoids to improve the health of aging males, further studies, especially long-term animal studies and clinical studies, are needed. Two of the important considerations in the studies are mechanism and efficacy of the flavonoid-enhanced testosterone biosynthesis. The mechanisms reported for the inhibitory effects of flavonoids on COX2-dependent signaling are mostly based on the studies with the cells from various tissues. These mechanisms need to be verified with Leydig cells. In addition to the COX2 dependent signaling, other mechanisms might be involved in the flavonoid-enhanced StAR gene expression and testosterone production. Further studies on the mechanisms may improve the understanding of the steroidogenic effects of flavonoids. Regarding the efficacy, bioavailability is an important factor being discussed in the studies on flavonoids (Setchell et al., 2001; Setchell et al., 2002). Specifically in the flavonoid-enhanced StAR gene expression and testosterone production, the following factors have been discussed recently: 1) Biphasic effects of flavonoids on StAR gene expression. As mentioned above, some of the flavonoids are able to block L-type calcium channels. It was reported that blocking L-type calcium channel produced biphasic effects on StAR gene expression in Leydig cells (Pandey et al., 2010). While these flavonoids might enhance StAR gene expression by reducing the transcriptional repressor DAX-1 protein, they might inhibit the positive effect of calciumdependent signaling on StAR gene expression; 2) Inhibitory effects of flavonoids on steroidogenic enzymes. It was reported that some of the flavonoids are able to inhibit activities of the enzymes involved in testosterone biosynthesis (Figueiroa et al., 2009; Hu et al., 2010). These inhibitory effects may reduce the efficacy of flavonoid-enhanced testosterone production; 3) Levels of cAMP in Leydig cells. The levels of cAMP-PKAphosphorylation are critical for the flavonoid-enhanced StAR gene expression in Leydig cells. As shown in Fig. 2C, in the absence of cAMP, flavonoid alone is not able to induce significant increases in StAR protein expression and steroid hormone production (Jana et al., 2008; Li et al., 2011). In the studies without exogenous cAMP, the endogenous cAMP becomes important for the steroidogenic effect of flavonoids. When the levels of endogenous cAMP or PKA activity in Leydig cells are not sufficient, flavonoid might not be able to induce significant increase in StAR gene expression. Therefore, minimal level of cAMP or PKA activity should be considered for the flavonoid-enhanced StAR gene expression and testosterone biosynthesis.
