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Bombyx E75 isoforms display stage- and tissue-specific responses to 20-hydroxyecdysone.

Li K, Guo E, Hossain MS, Li Q, Cao Y, Tian L, Deng X, Li S - Sci Rep (2015)

Bottom Line: At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E.At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues.In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

View Article: PubMed Central - PubMed

Affiliation: 1] Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding/Guangdong Provincial Sericulture and Mulberry Engineering Research Center, College of Animal Sciences, South China Agricultural University, Guangzhou, 510642, China [2] Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

ABSTRACT
Resulted from alternative splicing of the 5' exons, the nuclear receptor gene E75 in the silkworm, Bombyx mori, processes three mRNA isoforms, BmE75A, BmE75B and BmE75C. From the early 5(th) larval instar to the prepupal stages, BmE75A mRNA and protein levels in the prothoracic glands display developmental profiles similar to ecdysteroid titer. In the fat body, mRNA levels but not protein levels of all three BmE75 isoforms correlate with ecdysteroid titer; moreover, proteins of all three BmE75 isoforms disappear at the prepupal stages, and a modified BmE75 protein with smaller molecular weight and cytoplasm localization occurs. At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E. At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues. In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

No MeSH data available.


Responses of BmE75 isoforms to 20E in BmN cells.(A-A”) Responses of mRNA levels of BmE75A (A), BmE75B (A’) and BmE75C (A”) to 2 μM 20E or/and 10 μg/ml cycloheximide (CHX) in BmN cells. (B-B’) Response of protein levels of BmE75A, BmE75B and BmE75C to 20E or/and CHX in BmN cells 2 hours after treatment. (B’) Quantification of BmE75A, BmE75B and BmE75C protein levels in (B).
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f3: Responses of BmE75 isoforms to 20E in BmN cells.(A-A”) Responses of mRNA levels of BmE75A (A), BmE75B (A’) and BmE75C (A”) to 2 μM 20E or/and 10 μg/ml cycloheximide (CHX) in BmN cells. (B-B’) Response of protein levels of BmE75A, BmE75B and BmE75C to 20E or/and CHX in BmN cells 2 hours after treatment. (B’) Quantification of BmE75A, BmE75B and BmE75C protein levels in (B).

Mentions: To examine how BmE75 isoforms respond to 20E, we treated ovary-derived Bombyx BmN cells with 20E in the presence or absence of a protein synthesis inhibitor, cycloheximide (CHX). As determined by quantitative real-time PCR (qPCR), mRNA levels of BmE75A and BmE75B increased approximately 15-fold 1 hour after 20E treatment, no matter whether CHX was present or absent (Figs. 3A, A’). BmE75A mRNA level continued increasing to approximately 100-fold 2 hours after 20E treatment and the increase slightly dropped at the next hours (Fig. 3A). Meanwhile, BmE75B mRNA level continued increasing to approximately 60-fold 4 hours after 20E treatment and the increase slightly dropped at the next hours (Fig. 3A’). Importantly, CHX did not affect expression of BmE75A and BmE75B; moreover, the 20E-induced expression of BmE75A and BmE75B was not blocked by CHX, indicating that both isoforms are 20E primary-responsive in BmN cells (Figs. 3A, A’). By contrast, mRNA level of BmE75C did not change 1 hour after 20E treatment, increased only 2-fold 2 and 4 hours after 20E treatment, and decreased to the initial level 8 hours after 20E treatment. Interestingly, despite that CHX also weakly induced BmE75C expression, CHX and 20E together did not show overlapping induction of BmE75C expression (Fig. 3A”). Overall, 20E rapidly and abundantly induces expression of BmE75A and BmE75B in BmN cells, while its induction of BmE75C expression is slow and weak, showing similar results observed in the Bombyx ovary22.


Bombyx E75 isoforms display stage- and tissue-specific responses to 20-hydroxyecdysone.

Li K, Guo E, Hossain MS, Li Q, Cao Y, Tian L, Deng X, Li S - Sci Rep (2015)

Responses of BmE75 isoforms to 20E in BmN cells.(A-A”) Responses of mRNA levels of BmE75A (A), BmE75B (A’) and BmE75C (A”) to 2 μM 20E or/and 10 μg/ml cycloheximide (CHX) in BmN cells. (B-B’) Response of protein levels of BmE75A, BmE75B and BmE75C to 20E or/and CHX in BmN cells 2 hours after treatment. (B’) Quantification of BmE75A, BmE75B and BmE75C protein levels in (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499807&req=5

f3: Responses of BmE75 isoforms to 20E in BmN cells.(A-A”) Responses of mRNA levels of BmE75A (A), BmE75B (A’) and BmE75C (A”) to 2 μM 20E or/and 10 μg/ml cycloheximide (CHX) in BmN cells. (B-B’) Response of protein levels of BmE75A, BmE75B and BmE75C to 20E or/and CHX in BmN cells 2 hours after treatment. (B’) Quantification of BmE75A, BmE75B and BmE75C protein levels in (B).
Mentions: To examine how BmE75 isoforms respond to 20E, we treated ovary-derived Bombyx BmN cells with 20E in the presence or absence of a protein synthesis inhibitor, cycloheximide (CHX). As determined by quantitative real-time PCR (qPCR), mRNA levels of BmE75A and BmE75B increased approximately 15-fold 1 hour after 20E treatment, no matter whether CHX was present or absent (Figs. 3A, A’). BmE75A mRNA level continued increasing to approximately 100-fold 2 hours after 20E treatment and the increase slightly dropped at the next hours (Fig. 3A). Meanwhile, BmE75B mRNA level continued increasing to approximately 60-fold 4 hours after 20E treatment and the increase slightly dropped at the next hours (Fig. 3A’). Importantly, CHX did not affect expression of BmE75A and BmE75B; moreover, the 20E-induced expression of BmE75A and BmE75B was not blocked by CHX, indicating that both isoforms are 20E primary-responsive in BmN cells (Figs. 3A, A’). By contrast, mRNA level of BmE75C did not change 1 hour after 20E treatment, increased only 2-fold 2 and 4 hours after 20E treatment, and decreased to the initial level 8 hours after 20E treatment. Interestingly, despite that CHX also weakly induced BmE75C expression, CHX and 20E together did not show overlapping induction of BmE75C expression (Fig. 3A”). Overall, 20E rapidly and abundantly induces expression of BmE75A and BmE75B in BmN cells, while its induction of BmE75C expression is slow and weak, showing similar results observed in the Bombyx ovary22.

Bottom Line: At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E.At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues.In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

View Article: PubMed Central - PubMed

Affiliation: 1] Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding/Guangdong Provincial Sericulture and Mulberry Engineering Research Center, College of Animal Sciences, South China Agricultural University, Guangzhou, 510642, China [2] Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

ABSTRACT
Resulted from alternative splicing of the 5' exons, the nuclear receptor gene E75 in the silkworm, Bombyx mori, processes three mRNA isoforms, BmE75A, BmE75B and BmE75C. From the early 5(th) larval instar to the prepupal stages, BmE75A mRNA and protein levels in the prothoracic glands display developmental profiles similar to ecdysteroid titer. In the fat body, mRNA levels but not protein levels of all three BmE75 isoforms correlate with ecdysteroid titer; moreover, proteins of all three BmE75 isoforms disappear at the prepupal stages, and a modified BmE75 protein with smaller molecular weight and cytoplasm localization occurs. At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E. At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues. In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

No MeSH data available.