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- Afrakhte, Mozhgan, et al.
(författare)
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Induction of inhibitory Smad6 and Smad7 mRNA by TGF-beta family members
- 1998
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 249:2, s. 505-11
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Tidskriftsartikel (refereegranskat)abstract
- Smad6 and Smad7 function as intracellular antagonists in transforming growth factor-beta (TGF-beta) signaling. Here we report the isolation of human Smad6, which is closely related to Smad7. Smad6 and Smad7 mRNAs were differentially expressed in lung cancer cell lines and were rapidly and directly induced by TGF-beta1, activin and bone morphogenetic protein-7. Cross-talk between TGF-beta and other signaling pathways was demonstrated by the finding that epidermal growth factor (EGF) induced the expression of inhibitory SMAD mRNA. Moreover, whereas the phorbol ester PMA alone had no effect, it potentiated the TGF-beta1-induced expression of Smad7 mRNA. Ectopic expression of anti-sense Smad7 RNA was found to increase the effect of TGF-beta1, supporting its role as a negative regulator in TGF-beta signaling. Thus, expression of inhibitory Smads is induced by multiple stimuli, including the various TGF-beta family members, whose action they antagonize.
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| 2. |
- Mälarstig, Anders, et al.
(författare)
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A quantitative real-time PCR method for tissue factor mRNA
- 2003
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Ingår i: Thrombosis Research. - : Elsevier BV. - 0049-3848 .- 1879-2472. ; 112:3, s. 175-83
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Tissue factor (TF) is primarily known for its function to initiate blood coagulation. The range of in vivo expression of TF is wide and requires a dynamic assay for monitoring. A general method for TF mRNA quantitation that is dynamic, sensitive and applicable to a variety of experimental systems or clinical situations is therefore desirable. OBJECTIVES: To develop a method for sensitive and dynamic quantitation of TF mRNA in human blood cells. METHODS: TF mRNA expression was analysed and evaluated in monocyte isolations, in whole blood (healthy volunteers and patients scheduled for percutaneous coronary intervention, PCI) and in a panel of human cell lines. RNA was extracted, reverse transcribed and subjected to real-time PCR amplification, according to the TaqMan technology. A TF plasmid was constructed as calibrator of the assay. Two housekeeping genes used as endogenous controls for cDNA quality and integrity were evaluated. RESULTS: The assay was linear by seven orders of magnitude and detected down to 10(2) copies of the TF plasmid. The coefficient of variation was 4% intra-assay and 28% between the assays when using beta2MG as endogenous control. The beta-actin gene expression was induced by treatment with lipopolysaccharide (LPS) in blood leukocytes and could not be used as an endogenous control. However, beta2MG showed only minor variations upon treatment with LPS. The TF mRNA and antigen expression, measured in a Western blot, correlated well (R(2)=0.903) in a panel of 11 human cell lines. CONCLUSIONS: We have established a method for sensitive and dynamic quantitation of TF mRNA in experimental systems and for clinical situations.
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