Related Experiment Videos
Retinoic acid stimulates protein kinase A-associated G proteins during human teratocarcinoma differentiation
J M Kurie1, J Allopenna, E Dmitrovsky
1Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Abstract:
Retinoic acid (RA) treatment of F9 murine teratocarcinoma (TC) cells reduces the expression of the protein kinase A (PKA)-associated G protein, G alpha i2. The present study reveals interactions between the RA and PKA pathways during differentiation of the multipotent human TC cell line NTERA-2 clone D1 (abbreviated NT2/D1) which differ from prior reports in F9 TC cells. Compared to untreated NT2/D1 cells, differentiated NT2/D1 cells expressed increased levels of G alpha s and G alpha i1,2 proteins as shown by both immunoblot analysis and cholera toxin- and pertussis toxin-induced ADP ribosylation. To further explore cooperation between these pathways during human TC differentiation, we examined the effects of cyclic adenosine monophosphate (cAMP) on RA-responsive genes and of RA treatment on the transcriptional activation of a cAMP response element (CRE). Compared to RA alone, combined treatment with RA and cAMP augmented the expression of the RA nuclear receptor-beta (RAR-beta). Also, transient transfection assays revealed that cAMP and RA cooperated to enhance CRE transcriptional activation. The cAMP-induced enhancement of RA actions in NT2/D1 cells extended to immunophenotypic changes typical of the neuronal differentiation program induced by RA. In contrast to these findings in NT2/D1 cells, prior work in F9 TC cells showed that cAMP inhibits the RA-mediated augmentation of RAR-beta expression and switches the differentiation program from visceral to parietal endoderm. Thus, unlike murine TC cells, in human NT2/D1 cells RA stimulates PKA-associated G proteins and PKA pathway activation enhances RA-mediated TC differentiation.
Insights
Retinoic acid (RA) and protein kinase A (PKA) pathways interact differently during human teratocarcinoma (TC) cell differentiation. In human cells, RA stimulates PKA, enhancing TC differentiation, unlike in mouse cells.
Area of Science:
- Cell Biology
- Developmental Biology
- Molecular Biology
Background:
- Retinoic acid (RA) is crucial for teratocarcinoma (TC) cell differentiation.
- Protein kinase A (PKA) signaling pathways are implicated in cellular differentiation processes.
- Previous studies in F9 murine TC cells indicated antagonistic interactions between RA and PKA signaling.
Purpose of the Study:
- To investigate the interactions between RA and PKA pathways during human TC cell differentiation.
- To compare these interactions with those observed in murine TC cells.
- To elucidate the role of PKA pathway activation in RA-mediated human TC differentiation.
Main Methods:
- Immunoblot analysis to detect G protein levels.
- Cholera toxin and pertussis toxin-induced ADP ribosylation assays.
- Transient transfection assays to assess transcriptional activation.
- Analysis of RA nuclear receptor-beta (RAR-beta) expression.
- Assessment of immunophenotypic changes related to neuronal differentiation.
Main Results:
- Human NT2/D1 TC cells showed increased G alpha s and G alpha i1,2 protein levels upon differentiation.
- Combined RA and cyclic adenosine monophosphate (cAMP) treatment augmented RAR-beta expression more than RA alone.
- cAMP and RA cooperated to enhance cAMP response element (CRE) transcriptional activation.
- PKA pathway activation enhanced RA-mediated neuronal differentiation in NT2/D1 cells.
- These findings contrast with inhibitory effects of cAMP on RA signaling in F9 murine TC cells.
Conclusions:
- In human NT2/D1 TC cells, RA stimulates PKA-associated G proteins.
- PKA pathway activation cooperates with RA to enhance human TC differentiation, particularly neuronal differentiation.
- This represents a significant divergence from the antagonistic interactions observed in murine TC cells.