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Identification of the primary growth response gene, ST2/T1, as a gene whose expression is differentially regulated by
A Kieser1, J Goodnight, W Kölch
1Molecular Genetics Section, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
Abstract:
Individual protein kinase C isozymes have been shown to play different roles in mediating proliferation, differentiation and transformation, but it is not known to what extent these effects involve induction of expression of particular genes. To explore the differential gene expression that might be induced by activation of different PKC isozymes, we stably transfected NIH 3T3 cells with expression vectors that encode the isozymes PKC-alpha, -beta II, -gamma, -delta, -epsilon, -sigma and -eta. Using differential display-reverse transcription-polymerase chain reaction we isolated a small cDNA that encodes a portion of the primary response gene, ST2 (also referred to as T1 or DER4), and we confirmed by RNA blot studies that ST2/T1 expression is differentially regulated by PKC isozymes. ST2/T1 mRNA is undetectable in the unstimulated parental NIH 3T3 cells that express only the alpha isozyme of PKC, but it can be induced by phorbol ester treatment. Clones that overexpress PKC-alpha, -delta or -epsilon similarly do not express ST2/T1 until they are stimulated with phorbol esters, which induces expression of ST2/T1 with kinetics similar to wild-type NIH 3T3 but to different extents. In contrast, ST2/T1 mRNA is already present in unstimulated cells that overexpress PKC-beta II, -gamma, -sigma and -eta, but phorbol ester greatly enhances ST2/T1 expression in these cells. These results suggest a differential role for PKC isozymes in mediating the ST2/T1 expression that is induced by growth stimuli.
Insights
Different protein kinase C (PKC) isozymes differentially regulate the ST2 gene. Some PKC isozymes require activation for ST2 gene expression, while others maintain basal ST2 expression that is enhanced by activation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Protein Kinase C (PKC) isozymes mediate cellular processes like proliferation and differentiation.
- The role of specific PKC isozymes in inducing gene expression is not fully understood.
Purpose of the Study:
- To investigate the differential gene expression induced by various PKC isozymes.
- To determine how different PKC isozymes regulate the expression of the ST2 gene.
Main Methods:
- Stable transfection of NIH 3T3 cells with vectors encoding different PKC isozymes.
- Differential display-reverse transcription-polymerase chain reaction (DD-RT-PCR) to identify differentially expressed genes.
- RNA blot analysis to confirm ST2/T1 gene regulation.
Main Results:
- ST2/T1 mRNA expression is differentially regulated by PKC isozymes.
- Cells expressing PKC-alpha, -delta, or -epsilon require phorbol ester stimulation for ST2/T1 induction.
- Cells overexpressing PKC-beta II, -gamma, -sigma, or -eta exhibit basal ST2/T1 expression, enhanced by phorbol ester.
Conclusions:
- PKC isozymes play distinct roles in mediating ST2/T1 gene expression.
- The regulation of ST2/T1 by PKC isozymes is dependent on the specific isozyme involved.