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Dynamic resolution of functionally related gene sets in response to acute heat stress.

Szustakowski JD, Kosinski PA, Marrese CA, Lee JH, Elliman SJ, Nirmala N, Kemp DM - BMC Mol. Biol. (2007)

Bottom Line: Protein turnover-related pathways were significantly affected including protein folding, pre-mRNA processing, mRNA splicing, proteolysis and proteasome-related pathways.Many responses were transient, tending to normalize within 24 hours.In summary, we show that the transcriptional response to acute cell stress is largely transient and proteosome-centric.

View Article: PubMed Central - HTML - PubMed

Affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, USA. joseph.szustakowski@novartis.com <joseph.szustakowski@novartis.com>

ABSTRACT

Background: Using a gene clustering strategy we determined intracellular pathway relationships within skeletal myotubes in response to an acute heat stress stimuli. Following heat shock, the transcriptome was analyzed by microarray in a temporal fashion to characterize the dynamic relationship of signaling pathways.

Results: Bioinformatics analyses exposed coordination of functionally-related gene sets, depicting mechanism-based responses to heat shock. Protein turnover-related pathways were significantly affected including protein folding, pre-mRNA processing, mRNA splicing, proteolysis and proteasome-related pathways. Many responses were transient, tending to normalize within 24 hours.

Conclusion: In summary, we show that the transcriptional response to acute cell stress is largely transient and proteosome-centric.

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Related in: MedlinePlus

C2C12 cells recover from heat shock with a characteristic profile of injury response. Temporal gene expression profiles of heat shocked C2C12 myotubes (47°C for 30 minutes). Heat shock treatment was abated at 0 hours and cells were restored to growth conditions. mRNA was isolated at 1, 2, 4, 8 and 24 hours post-heat shock. (A-D) Transcriptional activity of the indicated genes was measured using Affymetrix GeneChips and data was analyzed using GeneSpring bioinformatics software. Data points represent the mean of three independent studies (± S.D). (E-G) C2C12 cells stably expressing Foxo3a-GFP were differentiated into myotubes (E), heat shocked and recovered for 3 hours (F) or treated with 20 μM LY294002 for 3 hours (G). Cells were then viewed under fluorescent light to observe localization of the protein.
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Figure 1: C2C12 cells recover from heat shock with a characteristic profile of injury response. Temporal gene expression profiles of heat shocked C2C12 myotubes (47°C for 30 minutes). Heat shock treatment was abated at 0 hours and cells were restored to growth conditions. mRNA was isolated at 1, 2, 4, 8 and 24 hours post-heat shock. (A-D) Transcriptional activity of the indicated genes was measured using Affymetrix GeneChips and data was analyzed using GeneSpring bioinformatics software. Data points represent the mean of three independent studies (± S.D). (E-G) C2C12 cells stably expressing Foxo3a-GFP were differentiated into myotubes (E), heat shocked and recovered for 3 hours (F) or treated with 20 μM LY294002 for 3 hours (G). Cells were then viewed under fluorescent light to observe localization of the protein.

Mentions: In the present study, we exposed C2C12 skeletal myotubes to heat shock-induced damage in order to identify coordinate patterns of transcriptional regulation. To initially authenticate this cell-based injury model, we identified several stress-related biomarkers and regulatory patterns to confirm bona fide damage response in the cells. Following heat shock treatment, cells recovered in growth medium at 37°C, and gene expression profiles were generated at 0, 1, 2, 4, 8 and 24 hours post-treatment. ATF3 and c-jun are critically regulated in the neuronal response to damage and during degeneration. Furthermore, these genes may play an anti-apoptotic role in the survival response of neuronal cells [11-13]. Here, both ATF3 and c-jun transcript levels were upregulated in response to heat-shock, consistent with their reported coordinate role in cell damage response (Figure 1A). A transient peak in expression of both genes occurred at 8 hours and attenuated at 24 h post-damage induction, indicating a regulated damage-induced response. Vimentin, an intermediate filament protein that plays a role in wound healing [14] and response to thermal stress [15,16], was augmented immediately following heat shock, and peaked at 2 hours. This rapid response to cell stress was consistent with the role of vimentin in protection against cytotoxic effects (Figure 1B). Also, the membrane type 1-matrix metalloproteases MMP14 and MMP2, which are functional partners during skeletal development [17] and stress response in the context of vascular injury [18], were robustly co-regulated at the transcriptional level in response to heat shock of C2C12 skeletal myotubes. Their expression level rose within 2–4 hours of insult, followed by a repression of expression levels at 8 and 24 hours (Figure 1C).


Dynamic resolution of functionally related gene sets in response to acute heat stress.

Szustakowski JD, Kosinski PA, Marrese CA, Lee JH, Elliman SJ, Nirmala N, Kemp DM - BMC Mol. Biol. (2007)

C2C12 cells recover from heat shock with a characteristic profile of injury response. Temporal gene expression profiles of heat shocked C2C12 myotubes (47°C for 30 minutes). Heat shock treatment was abated at 0 hours and cells were restored to growth conditions. mRNA was isolated at 1, 2, 4, 8 and 24 hours post-heat shock. (A-D) Transcriptional activity of the indicated genes was measured using Affymetrix GeneChips and data was analyzed using GeneSpring bioinformatics software. Data points represent the mean of three independent studies (± S.D). (E-G) C2C12 cells stably expressing Foxo3a-GFP were differentiated into myotubes (E), heat shocked and recovered for 3 hours (F) or treated with 20 μM LY294002 for 3 hours (G). Cells were then viewed under fluorescent light to observe localization of the protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1904231&req=5

Figure 1: C2C12 cells recover from heat shock with a characteristic profile of injury response. Temporal gene expression profiles of heat shocked C2C12 myotubes (47°C for 30 minutes). Heat shock treatment was abated at 0 hours and cells were restored to growth conditions. mRNA was isolated at 1, 2, 4, 8 and 24 hours post-heat shock. (A-D) Transcriptional activity of the indicated genes was measured using Affymetrix GeneChips and data was analyzed using GeneSpring bioinformatics software. Data points represent the mean of three independent studies (± S.D). (E-G) C2C12 cells stably expressing Foxo3a-GFP were differentiated into myotubes (E), heat shocked and recovered for 3 hours (F) or treated with 20 μM LY294002 for 3 hours (G). Cells were then viewed under fluorescent light to observe localization of the protein.
Mentions: In the present study, we exposed C2C12 skeletal myotubes to heat shock-induced damage in order to identify coordinate patterns of transcriptional regulation. To initially authenticate this cell-based injury model, we identified several stress-related biomarkers and regulatory patterns to confirm bona fide damage response in the cells. Following heat shock treatment, cells recovered in growth medium at 37°C, and gene expression profiles were generated at 0, 1, 2, 4, 8 and 24 hours post-treatment. ATF3 and c-jun are critically regulated in the neuronal response to damage and during degeneration. Furthermore, these genes may play an anti-apoptotic role in the survival response of neuronal cells [11-13]. Here, both ATF3 and c-jun transcript levels were upregulated in response to heat-shock, consistent with their reported coordinate role in cell damage response (Figure 1A). A transient peak in expression of both genes occurred at 8 hours and attenuated at 24 h post-damage induction, indicating a regulated damage-induced response. Vimentin, an intermediate filament protein that plays a role in wound healing [14] and response to thermal stress [15,16], was augmented immediately following heat shock, and peaked at 2 hours. This rapid response to cell stress was consistent with the role of vimentin in protection against cytotoxic effects (Figure 1B). Also, the membrane type 1-matrix metalloproteases MMP14 and MMP2, which are functional partners during skeletal development [17] and stress response in the context of vascular injury [18], were robustly co-regulated at the transcriptional level in response to heat shock of C2C12 skeletal myotubes. Their expression level rose within 2–4 hours of insult, followed by a repression of expression levels at 8 and 24 hours (Figure 1C).

Bottom Line: Protein turnover-related pathways were significantly affected including protein folding, pre-mRNA processing, mRNA splicing, proteolysis and proteasome-related pathways.Many responses were transient, tending to normalize within 24 hours.In summary, we show that the transcriptional response to acute cell stress is largely transient and proteosome-centric.

View Article: PubMed Central - HTML - PubMed

Affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, USA. joseph.szustakowski@novartis.com <joseph.szustakowski@novartis.com>

ABSTRACT

Background: Using a gene clustering strategy we determined intracellular pathway relationships within skeletal myotubes in response to an acute heat stress stimuli. Following heat shock, the transcriptome was analyzed by microarray in a temporal fashion to characterize the dynamic relationship of signaling pathways.

Results: Bioinformatics analyses exposed coordination of functionally-related gene sets, depicting mechanism-based responses to heat shock. Protein turnover-related pathways were significantly affected including protein folding, pre-mRNA processing, mRNA splicing, proteolysis and proteasome-related pathways. Many responses were transient, tending to normalize within 24 hours.

Conclusion: In summary, we show that the transcriptional response to acute cell stress is largely transient and proteosome-centric.

Show MeSH
Related in: MedlinePlus