Ulysses
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Cyclooxygenase-2 Regulation of the Age-Related Decline in Testosterone Biosynthesis | Endocrinology | Oxford Academic
...The age-related decline in testosterone biosynthesis in testicular Leydig cells has been well documented, but the mechanisms involved in the decline are not clear. Recent studies have described a cyclooxygenase-2 (COX2)-dependent tonic inhibition of Leydig cell steroidogenesis and expression of the steroidogenic acute regulatory protein (StAR). The present study was conducted to determine whether COX2 protein increases with age in rat Leydig cells and whether COX2 plays a role in the age-related decline in testosterone biosynthesis. Our results indicate that from 3 months of age to 30 months, COX2 protein in aged rat Leydig cells increased by 346% over that of young Leydig cells, StAR protein decreased to 33%, and blood testosterone concentration and testosterone biosynthesis in Leydig cells decreased to 41 and 33%, respectively. Further experiments demonstrated that overexpressing COX2 in MA-10 mouse Leydig cells inhibited StAR gene expression and steroidogenesis and that the inhibitory effects of COX2 could be reversed by blocking COX2 activity. Notably, incubation of aged Leydig cells with the COX2 inhibitor NS398 enhanced their testosterone biosynthesis. Blood testosterone concentrations in aged rats fed the COX2 inhibitor DFU, at doses of 5, 10, 15, and 20 mg/kg body weight per day were increased by 15, 23, 56, and 120%, respectively, over the levels in the rats receiving no DFU. The present study suggests a novel mechanism in male aging involving COX2 and a potential application of the mechanism to delay the age-related decline in testosterone biosynthesis.
...StAR gene expression and testosterone biosynthesis are regulated by LH released from the pituitary gland. LH stimulation of Leydig cells induces cAMP formation, which activates protein kinase A (PKA). PKA in turn phosphorylates transcription factors that regulate StAR gene expression. LH and cAMP also stimulate arachidonic acid (AA) release (18–21), and AA metabolites transduce signals to the nucleus that are also involved in regulating StAR gene transcription (22). These two LH-activated signaling pathways act to coregulate StAR gene expression and steroidogenesis. Positive or negative changes in the signals in either pathway will dramatically affect StAR gene expression, which will subsequently affect steroid hormone biosynthesis in Leydig cells (22, 23). Regarding AA-mediated signaling, AA is primarily metabolized by the lipoxygenase, epoxygenase, or cyclooxygenase enzyme pathway. Whereas AA metabolites produced by lipoxygenase and epoxygenase activities may enhance steroid hormone biosynthesis (24), previous studies suggested that cyclooxygenase-2 (COX2), an isoform of cyclooxygenase, produces a tonic inhibition of StAR gene expression and steroidogenesis in Leydig cells (23). It has been reported that COX2 expression is up-regulated during the aging process in various tissues (25, 26). Also, using cDNA microarray screening, it has previously been demonstrated that COX2 mRNA levels were increased in aged Leydig cells (27), but a cause-and-effect relationship between COX2 and aging was not apparent in that study. Our reasoning was that if COX2 protein increases in aging Leydig cells, it is possible that the tonic inhibition caused by COX2 activity plays an important role in the age-related decrease in testosterone production. The present study sought to determine whether COX2 protein increases during the course of normal aging in Leydig cells and, if so, to investigate the impact of this increase on StAR gene expression and testosterone biosynthesis.
...It was found that inhibition of COX2 activity with DFU, as assessed by measuring prostaglandin E2 levels, increased blood testosterone concentrations (P < 0.05) and StAR protein in Leydig cells in a dose-dependent manner. Blood testosterone concentrations in the rats fed 5, 10, 15, and 20 mg DFU per kilogram body weight per day were increased by 15, 23, 56, and 120%, respectively, over the levels in rats receiving no DFU (Fig. 6).[/quote]
...To investigate the mechanism for the COX2-dependent tonic inhibition of StAR gene expression and steroidogenesis in Leydig cells, HPLC was employed to determine which AA metabolites in Leydig cells are affected by COX2 activity. The results in Fig. 7 show that inhibition of COX2 activity in MA-10 Leydig cells with 20 μm of the COX2 inhibitor NS398 significantly increased 5-HPETE (P < 0.05) and 5-HETE (P < 0.05). Both of these AA metabolites are derived through the action of 5-lipoxygenase and have been previously reported to enhance StAR gene expression and steroidogenesis (35). To determine whether a 5-lipoxygenase metabolite of AA is able to increase testosterone synthesis in aged Leydig cells, 5-HETE was added to cultured Leydig cells isolated from 24-month-old male Brown Norway rats, and then the cells were stimulated with human chorionic gonadotropin (hCG). As shown in Fig. 8, incubation with 5-HETE significantly increased hCG-stimulated testosterone production (P < 0.05).
...Whereas it is still unknown as to how COX2 achieves its inhibitory effect, taken together, our data indicate that a COX2-dependent tonic inhibition of StAR gene expression increases during the course of male aging and reduces the sensitivity of Leydig cells to LH or cAMP stimulation. Consequently, the age-related increase in COX2 expression results in a decline in testosterone biosynthesis. Because COX2 gene expression is up-regulated by many pathological and stress-related factors, including reactive oxygen (26, 36), the present study provides important information for understanding the roles of these factors in the aging process.
...Whereas other steroidogenic proteins could affect blood testosterone levels, our previous work suggested that the increased steroidogenesis resulting from inhibition of COX2 activity is mainly due to an increased cholesterol supply because there was no significant difference in steroid production among the experimental groups when 22(R)-hydroxycholesterol was used as substrate (23). This agrees with our results from experiments with isolated aged Leydig cells and also those with aged rats, in which the COX2 inhibitor-increased testosterone production is always concomitant with increased StAR protein, known to play a critical role in cholesterol supply.
...The age-related decline in testosterone biosynthesis in testicular Leydig cells has been well documented, but the mechanisms involved in the decline are not clear. Recent studies have described a cyclooxygenase-2 (COX2)-dependent tonic inhibition of Leydig cell steroidogenesis and expression of the steroidogenic acute regulatory protein (StAR). The present study was conducted to determine whether COX2 protein increases with age in rat Leydig cells and whether COX2 plays a role in the age-related decline in testosterone biosynthesis. Our results indicate that from 3 months of age to 30 months, COX2 protein in aged rat Leydig cells increased by 346% over that of young Leydig cells, StAR protein decreased to 33%, and blood testosterone concentration and testosterone biosynthesis in Leydig cells decreased to 41 and 33%, respectively. Further experiments demonstrated that overexpressing COX2 in MA-10 mouse Leydig cells inhibited StAR gene expression and steroidogenesis and that the inhibitory effects of COX2 could be reversed by blocking COX2 activity. Notably, incubation of aged Leydig cells with the COX2 inhibitor NS398 enhanced their testosterone biosynthesis. Blood testosterone concentrations in aged rats fed the COX2 inhibitor DFU, at doses of 5, 10, 15, and 20 mg/kg body weight per day were increased by 15, 23, 56, and 120%, respectively, over the levels in the rats receiving no DFU. The present study suggests a novel mechanism in male aging involving COX2 and a potential application of the mechanism to delay the age-related decline in testosterone biosynthesis.
...StAR gene expression and testosterone biosynthesis are regulated by LH released from the pituitary gland. LH stimulation of Leydig cells induces cAMP formation, which activates protein kinase A (PKA). PKA in turn phosphorylates transcription factors that regulate StAR gene expression. LH and cAMP also stimulate arachidonic acid (AA) release (18–21), and AA metabolites transduce signals to the nucleus that are also involved in regulating StAR gene transcription (22). These two LH-activated signaling pathways act to coregulate StAR gene expression and steroidogenesis. Positive or negative changes in the signals in either pathway will dramatically affect StAR gene expression, which will subsequently affect steroid hormone biosynthesis in Leydig cells (22, 23). Regarding AA-mediated signaling, AA is primarily metabolized by the lipoxygenase, epoxygenase, or cyclooxygenase enzyme pathway. Whereas AA metabolites produced by lipoxygenase and epoxygenase activities may enhance steroid hormone biosynthesis (24), previous studies suggested that cyclooxygenase-2 (COX2), an isoform of cyclooxygenase, produces a tonic inhibition of StAR gene expression and steroidogenesis in Leydig cells (23). It has been reported that COX2 expression is up-regulated during the aging process in various tissues (25, 26). Also, using cDNA microarray screening, it has previously been demonstrated that COX2 mRNA levels were increased in aged Leydig cells (27), but a cause-and-effect relationship between COX2 and aging was not apparent in that study. Our reasoning was that if COX2 protein increases in aging Leydig cells, it is possible that the tonic inhibition caused by COX2 activity plays an important role in the age-related decrease in testosterone production. The present study sought to determine whether COX2 protein increases during the course of normal aging in Leydig cells and, if so, to investigate the impact of this increase on StAR gene expression and testosterone biosynthesis.
...It was found that inhibition of COX2 activity with DFU, as assessed by measuring prostaglandin E2 levels, increased blood testosterone concentrations (P < 0.05) and StAR protein in Leydig cells in a dose-dependent manner. Blood testosterone concentrations in the rats fed 5, 10, 15, and 20 mg DFU per kilogram body weight per day were increased by 15, 23, 56, and 120%, respectively, over the levels in rats receiving no DFU (Fig. 6).[/quote]
...To investigate the mechanism for the COX2-dependent tonic inhibition of StAR gene expression and steroidogenesis in Leydig cells, HPLC was employed to determine which AA metabolites in Leydig cells are affected by COX2 activity. The results in Fig. 7 show that inhibition of COX2 activity in MA-10 Leydig cells with 20 μm of the COX2 inhibitor NS398 significantly increased 5-HPETE (P < 0.05) and 5-HETE (P < 0.05). Both of these AA metabolites are derived through the action of 5-lipoxygenase and have been previously reported to enhance StAR gene expression and steroidogenesis (35). To determine whether a 5-lipoxygenase metabolite of AA is able to increase testosterone synthesis in aged Leydig cells, 5-HETE was added to cultured Leydig cells isolated from 24-month-old male Brown Norway rats, and then the cells were stimulated with human chorionic gonadotropin (hCG). As shown in Fig. 8, incubation with 5-HETE significantly increased hCG-stimulated testosterone production (P < 0.05).
...Whereas it is still unknown as to how COX2 achieves its inhibitory effect, taken together, our data indicate that a COX2-dependent tonic inhibition of StAR gene expression increases during the course of male aging and reduces the sensitivity of Leydig cells to LH or cAMP stimulation. Consequently, the age-related increase in COX2 expression results in a decline in testosterone biosynthesis. Because COX2 gene expression is up-regulated by many pathological and stress-related factors, including reactive oxygen (26, 36), the present study provides important information for understanding the roles of these factors in the aging process.
...Whereas other steroidogenic proteins could affect blood testosterone levels, our previous work suggested that the increased steroidogenesis resulting from inhibition of COX2 activity is mainly due to an increased cholesterol supply because there was no significant difference in steroid production among the experimental groups when 22(R)-hydroxycholesterol was used as substrate (23). This agrees with our results from experiments with isolated aged Leydig cells and also those with aged rats, in which the COX2 inhibitor-increased testosterone production is always concomitant with increased StAR protein, known to play a critical role in cholesterol supply.
