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Ornithine Decarboxylase Activity Is Required for Prostatic Budding in the Developing Mouse Prostate.

Gamat M, Malinowski RL, Parkhurst LJ, Steinke LM, Marker PC - PLoS ONE (2015)

Bottom Line: Inhibiting ornithine decarboxylase using DFMO in UGS organ culture blocked the induction of prostatic buds by androgens, and significantly decreased expression of key prostate transcription factor, Nkx3.1, by androgens.DFMO also significantly decreased the expression of developmental regulatory gene Notch1.Together these results indicate that Odc1 and polyamines are required for androgens to exert their effect in mediating prostatic bud induction, and are required for the expression of a subset of prostatic developmental regulatory genes including Notch1 and Nkx3.1.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, University of Wisconsin-Madison, 777 Highland Avenue, Madison, WI, United States of America.

ABSTRACT
The prostate is a male accessory sex gland that produces secretions in seminal fluid to facilitate fertilization. Prostate secretory function is dependent on androgens, although the mechanism by which androgens exert their effects is still unclear. Polyamines are small cationic molecules that play pivotal roles in DNA transcription, translation and gene regulation. The rate-limiting enzyme in polyamine biosynthesis is ornithine decarboxylase, which is encoded by the gene Odc1. Ornithine decarboxylase mRNA decreases in the prostate upon castration and increases upon administration of androgens. Furthermore, testosterone administered to castrated male mice restores prostate secretory activity, whereas administering testosterone and the ornithine decarboxylase inhibitor D,L-α-difluromethylornithine (DFMO) to castrated males does not restore prostate secretory activity, suggesting that polyamines are required for androgens to exert their effects. To date, no one has examined polyamines in prostate development, which is also androgen dependent. In this study, we showed that ornithine decarboxylase protein was expressed in the epithelium of the ventral, dorsolateral and anterior lobes of the adult mouse prostate. Ornithine decarboxylase protein was also expressed in the urogenital sinus (UGS) epithelium of the male and female embryo prior to prostate development, and expression continued in prostatic epithelial buds as they emerged from the UGS. Inhibiting ornithine decarboxylase using DFMO in UGS organ culture blocked the induction of prostatic buds by androgens, and significantly decreased expression of key prostate transcription factor, Nkx3.1, by androgens. DFMO also significantly decreased the expression of developmental regulatory gene Notch1. Other genes implicated in prostatic development including Sox9, Wif1 and Srd5a2 were unaffected by DFMO. Together these results indicate that Odc1 and polyamines are required for androgens to exert their effect in mediating prostatic bud induction, and are required for the expression of a subset of prostatic developmental regulatory genes including Notch1 and Nkx3.1.

No MeSH data available.


Related in: MedlinePlus

Allowing prostatic buds to grow before polyamine depletion did not rescue Nkx3.1 expression.We determined the time course of prostatic bud formation over 2 days of culture in testosterone, by culturing the UGS in the presence of testosterone for the time indicated, fixed the cultures, and stained them with E-Cadherin to examine prostatic bud formation. After 24 hours or 1 day in culture, prostatic buds were still not apparent (A). After 1.5 days in testosterone, several prostatic buds were observed (B, white arrowheads). The buds were longer and more numerous after 2 days in testosterone (C, white arrowheads). Culturing the UGS in testosterone for two days to allow buds to grow, followed by polyamine depletion did not rescue Nkx3.1 expression (D). *, p<0.05.
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pone.0139522.g008: Allowing prostatic buds to grow before polyamine depletion did not rescue Nkx3.1 expression.We determined the time course of prostatic bud formation over 2 days of culture in testosterone, by culturing the UGS in the presence of testosterone for the time indicated, fixed the cultures, and stained them with E-Cadherin to examine prostatic bud formation. After 24 hours or 1 day in culture, prostatic buds were still not apparent (A). After 1.5 days in testosterone, several prostatic buds were observed (B, white arrowheads). The buds were longer and more numerous after 2 days in testosterone (C, white arrowheads). Culturing the UGS in testosterone for two days to allow buds to grow, followed by polyamine depletion did not rescue Nkx3.1 expression (D). *, p<0.05.

Mentions: We asked whether the reduction in Nkx3.1 is due to the lack of prostatic buds, or whether it is due to a direct effect of polyamine depletion. To do this, we cultured the UGS in testosterone until prostatic buds were first morphologically identifiable, then depleted polyamines using DFMO. Firstly, we determined the period of time required for prostatic buds to become morphologically distinct. Even after six days of culture in no steroid treatment, we did not observe any prostatic buds (Fig 4A). After day 1 of culture in testosterone, we also did not observe prostatic buds (8A). The first morphological buds were distinguished at day 1.5 of culture in testosterone (Fig 8B, white arrowheads) with more buds visible at day 2 of culture in testosterone (Fig 8C, white arrowheads). We then cultured the UGS for two days in testosterone only to allow the prostatic buds to form, followed by polyamine depletion using DFMO for the next four days. Similar to our previous results, Nkx3.1 was significantly up-regulated after 6 days in testosterone compared to NS treatment (Fig 8D, p<0.05) and significantly down-regulated after 6 days in T+DFMO compared to testosterone alone (Fig 8D, p<0.05). However, culturing the UGS in testosterone for two days after prostatic buds have formed, followed by polyamine depletion did not rescue Nkx3.1, with Nkx3.1 significantly decreased after 2 days of testosterone + 4 days of T+DFMO, compared to testosterone alone (Fig 8D, p<0.05) and no significant difference between 6 days T+DFMO and 2 days T and 4 days T+DFMO. Although inhibiting ornithine decarboxylase after prostatic buds have formed did block Nkx3.1 expression, it did not reduce Notch1 expression (data not shown). Together, these results suggest that the DFMO-induced reduction of Nkx3.1 was not an indirect effect due to the loss of prostatic buds and that polyamines are directly required to maintain Nkx3.1 expression regardless of the presence or absence of buds.


Ornithine Decarboxylase Activity Is Required for Prostatic Budding in the Developing Mouse Prostate.

Gamat M, Malinowski RL, Parkhurst LJ, Steinke LM, Marker PC - PLoS ONE (2015)

Allowing prostatic buds to grow before polyamine depletion did not rescue Nkx3.1 expression.We determined the time course of prostatic bud formation over 2 days of culture in testosterone, by culturing the UGS in the presence of testosterone for the time indicated, fixed the cultures, and stained them with E-Cadherin to examine prostatic bud formation. After 24 hours or 1 day in culture, prostatic buds were still not apparent (A). After 1.5 days in testosterone, several prostatic buds were observed (B, white arrowheads). The buds were longer and more numerous after 2 days in testosterone (C, white arrowheads). Culturing the UGS in testosterone for two days to allow buds to grow, followed by polyamine depletion did not rescue Nkx3.1 expression (D). *, p<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4591331&req=5

pone.0139522.g008: Allowing prostatic buds to grow before polyamine depletion did not rescue Nkx3.1 expression.We determined the time course of prostatic bud formation over 2 days of culture in testosterone, by culturing the UGS in the presence of testosterone for the time indicated, fixed the cultures, and stained them with E-Cadherin to examine prostatic bud formation. After 24 hours or 1 day in culture, prostatic buds were still not apparent (A). After 1.5 days in testosterone, several prostatic buds were observed (B, white arrowheads). The buds were longer and more numerous after 2 days in testosterone (C, white arrowheads). Culturing the UGS in testosterone for two days to allow buds to grow, followed by polyamine depletion did not rescue Nkx3.1 expression (D). *, p<0.05.
Mentions: We asked whether the reduction in Nkx3.1 is due to the lack of prostatic buds, or whether it is due to a direct effect of polyamine depletion. To do this, we cultured the UGS in testosterone until prostatic buds were first morphologically identifiable, then depleted polyamines using DFMO. Firstly, we determined the period of time required for prostatic buds to become morphologically distinct. Even after six days of culture in no steroid treatment, we did not observe any prostatic buds (Fig 4A). After day 1 of culture in testosterone, we also did not observe prostatic buds (8A). The first morphological buds were distinguished at day 1.5 of culture in testosterone (Fig 8B, white arrowheads) with more buds visible at day 2 of culture in testosterone (Fig 8C, white arrowheads). We then cultured the UGS for two days in testosterone only to allow the prostatic buds to form, followed by polyamine depletion using DFMO for the next four days. Similar to our previous results, Nkx3.1 was significantly up-regulated after 6 days in testosterone compared to NS treatment (Fig 8D, p<0.05) and significantly down-regulated after 6 days in T+DFMO compared to testosterone alone (Fig 8D, p<0.05). However, culturing the UGS in testosterone for two days after prostatic buds have formed, followed by polyamine depletion did not rescue Nkx3.1, with Nkx3.1 significantly decreased after 2 days of testosterone + 4 days of T+DFMO, compared to testosterone alone (Fig 8D, p<0.05) and no significant difference between 6 days T+DFMO and 2 days T and 4 days T+DFMO. Although inhibiting ornithine decarboxylase after prostatic buds have formed did block Nkx3.1 expression, it did not reduce Notch1 expression (data not shown). Together, these results suggest that the DFMO-induced reduction of Nkx3.1 was not an indirect effect due to the loss of prostatic buds and that polyamines are directly required to maintain Nkx3.1 expression regardless of the presence or absence of buds.

Bottom Line: Inhibiting ornithine decarboxylase using DFMO in UGS organ culture blocked the induction of prostatic buds by androgens, and significantly decreased expression of key prostate transcription factor, Nkx3.1, by androgens.DFMO also significantly decreased the expression of developmental regulatory gene Notch1.Together these results indicate that Odc1 and polyamines are required for androgens to exert their effect in mediating prostatic bud induction, and are required for the expression of a subset of prostatic developmental regulatory genes including Notch1 and Nkx3.1.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, University of Wisconsin-Madison, 777 Highland Avenue, Madison, WI, United States of America.

ABSTRACT
The prostate is a male accessory sex gland that produces secretions in seminal fluid to facilitate fertilization. Prostate secretory function is dependent on androgens, although the mechanism by which androgens exert their effects is still unclear. Polyamines are small cationic molecules that play pivotal roles in DNA transcription, translation and gene regulation. The rate-limiting enzyme in polyamine biosynthesis is ornithine decarboxylase, which is encoded by the gene Odc1. Ornithine decarboxylase mRNA decreases in the prostate upon castration and increases upon administration of androgens. Furthermore, testosterone administered to castrated male mice restores prostate secretory activity, whereas administering testosterone and the ornithine decarboxylase inhibitor D,L-α-difluromethylornithine (DFMO) to castrated males does not restore prostate secretory activity, suggesting that polyamines are required for androgens to exert their effects. To date, no one has examined polyamines in prostate development, which is also androgen dependent. In this study, we showed that ornithine decarboxylase protein was expressed in the epithelium of the ventral, dorsolateral and anterior lobes of the adult mouse prostate. Ornithine decarboxylase protein was also expressed in the urogenital sinus (UGS) epithelium of the male and female embryo prior to prostate development, and expression continued in prostatic epithelial buds as they emerged from the UGS. Inhibiting ornithine decarboxylase using DFMO in UGS organ culture blocked the induction of prostatic buds by androgens, and significantly decreased expression of key prostate transcription factor, Nkx3.1, by androgens. DFMO also significantly decreased the expression of developmental regulatory gene Notch1. Other genes implicated in prostatic development including Sox9, Wif1 and Srd5a2 were unaffected by DFMO. Together these results indicate that Odc1 and polyamines are required for androgens to exert their effect in mediating prostatic bud induction, and are required for the expression of a subset of prostatic developmental regulatory genes including Notch1 and Nkx3.1.

No MeSH data available.


Related in: MedlinePlus