3.3 INTEGRACIÓN DE LOS SISTEMAS EN LA GESTIÓN DE LA
3.3.2 ANÁLISIS DE LAS DIFERENTES FUENTES TECNOLÓGICAS DE
Much is known about the mechanisms which regulate TNF-a-induced expression of inflammatory response marker proteins such as ICAM-1, VCAM-1, and lL-8. Transcription of these proteins downstream of TN F-a signalling is dependent on NF-kB activation (Wallach et al., 1999). Briefly, TN F-a treatment causes activation of the 1k-B kinases which phosphorylate 1k-B, causing rapid 1k-B degradation which releases its inhibition of NF-kB. NF-kB is then able to translocate to the nucleus to induce transcription by binding to consensus promoter sequences. lCAM-2 is constitutively expressed in endothelial cells and protein levels are decreased by TN F-a stimulation (McLaughlin et al., 1998), conversely to lCAM-1. This result was confirmed here, but a transient increase in lCAM-2 protein levels was observed 2 hours after TN F-a stimulation. Interestingly, the same protein expression pattern was shown by RhoA. Analysis of mRNA levels showed that a transient increase in lCAM-2 mRNA levels occurred after 1 hour of TN F-a stimulation. Also RhoA mRNA levels increased during 4 hours of TN F-a stimulation, indicating that lCAM-2 and RhoA transcription may be regulated by similar mechanisms. Indeed, it was recently published that both lCAM-2 and RhoA are transcriptionally controlled by the transcription factor Erg in endothelial cells (McLaughlin et al., 2001). Erg was previously shown to regulate the transcription of lCAM-2 downstream of TN F-a stimulation of HUVECs. RhoA was identified as an Erg target gene by a combined approach of antisense oligonucleotides and differential gene expression. In fact, the protein expression data for RhoA presented here was used as a personal communication in the article by McLaughlin et al (2001).
Erg is a member of the Ets family of transcription factors which are involved in embryonic development, cellular transformation and inflammation. A role for lCAM-2 in inflammation has yet to be identified and as a result down-regulation of lCAM-2 by TN F-a stimulation is not unexpected. However, RhoA down-regulation by TN F-a cannot be explained quite so simplistically. TN F-a stimulation induces stress fibre formation and subsequent formation of intercellular gaps which could be important for leukocyte transmigration. Moreover, monocyte binding to HUVECs requires Rho- regulated receptor clustering and lymphocyte transmigration across brain endothelial cell
monolayers requires endothelial cell Rho activation (Adamson et al., 1999; Wojciak- Stothard et al., 1999). In light of this evidence, it is possible that down-regulation of RhoA is required to decrease the magnitude of the endothelial cytoskeletal response to TNF-o/leukocyte transmigration. Higher levels of total RhoA activity may cause irreparable damage to the endothelial monolayer.
However, TN F-a stimulation did not appear to induce any changes in RhoC mRNA levels. RhoC has been shown to have a critical role in metastasis of tumour cells (Clark et al., 2000). RhoC was found to be upregulated in métastasés derived from 2 tumour cell lines. Metastasis was inhibited by N19RhoA, which is likely to inhibit all Rho isoforms by titration of RhoGEFs, showing that Rho activity may be necessary, and RhoC sufficient, for métastasés in these tumour cell lines. Introduction of RhoC into cells induced a motile cytoskeletal phenotype, not typical of that induced by RhoA. Therefore it is likely that RhoC induces different cytoskeletal structures from the stress fibres induced by RhoA.
RhoE mRNA levels and protein levels appeared to follow the same trend as RhoA after TN F-a stimulation. RhoE expression in MDCK cells decreases stress fibre levels and increases cell migration speed (Guasch et al., 1998). It is reasonable to expect that when endothelial cells need to make stress fibres they would down-regulate levels of proteins, such as RhoE, which would inhibit their formation. RhoE has previously been shown to be upregulated in activated Rafl-transformed MDCK cells (Hansen et al., 2000). This up regulation appears to be crucial for multilayering of MDCK cells as introduction of activated RhoA reversed the transformed phenotype. RhoE has also been shown to be upregulated by UVB irradiation in kératinocytes (Murakami et al., 2001). As RhoE is only found in its GTP-bound form, it is possible that an important regulatory control of RhoE is expression level. However, there is also a need to test the expression levels of Rndl/2 as Rndl is known to decrease stress fibre levels.
As the results shown for mRNA levels of Rho family proteins are presented in their quantitative form, only the increase of RhoE after TN F-a stimulation was statistically significant. The Lightcycler technology is designed to be extremely sensitive to small changes in mRNA levels. However, the system is dependent upon the investigators’ ability to pipette 2.0 pi accurately for many repetitions. Moreover the investigator relies
upon the accuracy of spectrophotometry and serial dilutions to obtain aliquots with 62 copies/|xl (fmol of DNA) from PCR preparations. Variation between experiments means that it is important to generate 3 independent sets of mRNA. As there was only time to generate 2 sets of mRNA, the results show definite trends but many of these are not statistically significant.
In conclusion, TN F-a induces transcriptional changes in Rho proteins of HUVECs. It is likely that RhoA and ICAM-2 are co-regulated and it is also possible that RhoE may be controlled by similar mechanisms. As Erg I has been shown to regulate expression of RhoA and ICAM-2, using antisense to ablate Ergl expression could be used to investigate RhoE expression at the promoter level (McLaughlin et al., 2001).