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The level of CD147 expression correlates with cyclophilin-induced signalling and chemotaxis.

Trachtenberg A, Pushkarsky T, Heine S, Constant S, Brichacek B, Bukrinsky M - BMC Res Notes (2011)

Bottom Line: However, CD147 is not known to associate with signal transducing molecules, so other transmembrane proteins, such as proteoglycans, integrins, and CD98, were suggested as receptor or co-receptor for eCyp.Here, we manipulated CD147 expression levels on HeLa cells using RNAi and investigated the signalling and chemotactic responses to eCypA.Both Erk activation and chemotaxis correlated with the level of CD147 expression, with cells exhibiting low level expression being practically unresponsive to eCypA.

View Article: PubMed Central - HTML - PubMed

Affiliation: The George Washington University, Washington, DC 20037, USA. mtmmib@gwumc.edu.

ABSTRACT

Background: Previous studies identified CD147 as the chemotactic receptor on inflammatory leukocytes for extracellular cyclophilins (eCyp). However, CD147 is not known to associate with signal transducing molecules, so other transmembrane proteins, such as proteoglycans, integrins, and CD98, were suggested as receptor or co-receptor for eCyp. CD147 is ubiquitously expressed on many cell types, but relationship between the level of CD147 expression and cellular responses to eCyp has never been analyzed. Given the role of eCyp in pathogenesis of many diseases, it is important to know whether cellular responses to eCyp are regulated at the level of CD147 expression.

Results: Here, we manipulated CD147 expression levels on HeLa cells using RNAi and investigated the signalling and chemotactic responses to eCypA. Both Erk activation and chemotaxis correlated with the level of CD147 expression, with cells exhibiting low level expression being practically unresponsive to eCypA.

Conclusions: Our results provide the first demonstration of a chemotactic response of HeLa cells to eCypA, establish a correlation between the level of CD147 expression and the magnitude of cellular responses to eCypA, and indicate that CD147 may be a limiting factor in the receptor complex determining cyclophilin-induced Erk activation and cell migration.

No MeSH data available.


Related in: MedlinePlus

CD147 expression and chemotactic activity. HeLa-CD4 cells stably transfected with anti-CD147 siRNA-expressing vector or empty vector control were sorted according to the level of CD147 expression into low, medium and high (cells transfected with empty vector) expressing clones (A). Chemotactic activity of the clones in response to CypA (B) or SDF-1α (C) was measured as described in Materials and Methods. Chemotactic activity was analyzed in triplicate and results are presented as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001. Results are shown for one representative experiment out of two performed.
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Figure 1: CD147 expression and chemotactic activity. HeLa-CD4 cells stably transfected with anti-CD147 siRNA-expressing vector or empty vector control were sorted according to the level of CD147 expression into low, medium and high (cells transfected with empty vector) expressing clones (A). Chemotactic activity of the clones in response to CypA (B) or SDF-1α (C) was measured as described in Materials and Methods. Chemotactic activity was analyzed in triplicate and results are presented as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001. Results are shown for one representative experiment out of two performed.

Mentions: To manipulate CD147 expression levels on HeLa cells we used the RNAi knockdown approach. HeLa-CD4 (MAGI) cells were transfected with the pSuper-retro vector (OligoEngine, Seattle, WA) containing puromycin resistance gene and siRNA corresponding to the nucleotides 470-490 of CD147 mRNA. Puromycin resistant cells displayed lower concentrations of surface CD147 than parental cell line. The cells with the highest CD147 surface expression were removed using magnetic beads followed by flow cytometric cell sorting of remaining cells. Cell clones with decreased CD147 abundance were established from the remaining cell population. Cell clones with low and medium CD147 expression were selected for further analysis in comparison to original HeLa-CD4 cell clones transduced with an empty vector (high level CD147 expressors) (Figure 1A). Importantly, the level of expression changed after several passages of cells, making it necessary to verify the expression level before each experiment.


The level of CD147 expression correlates with cyclophilin-induced signalling and chemotaxis.

Trachtenberg A, Pushkarsky T, Heine S, Constant S, Brichacek B, Bukrinsky M - BMC Res Notes (2011)

CD147 expression and chemotactic activity. HeLa-CD4 cells stably transfected with anti-CD147 siRNA-expressing vector or empty vector control were sorted according to the level of CD147 expression into low, medium and high (cells transfected with empty vector) expressing clones (A). Chemotactic activity of the clones in response to CypA (B) or SDF-1α (C) was measured as described in Materials and Methods. Chemotactic activity was analyzed in triplicate and results are presented as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001. Results are shown for one representative experiment out of two performed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198701&req=5

Figure 1: CD147 expression and chemotactic activity. HeLa-CD4 cells stably transfected with anti-CD147 siRNA-expressing vector or empty vector control were sorted according to the level of CD147 expression into low, medium and high (cells transfected with empty vector) expressing clones (A). Chemotactic activity of the clones in response to CypA (B) or SDF-1α (C) was measured as described in Materials and Methods. Chemotactic activity was analyzed in triplicate and results are presented as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001. Results are shown for one representative experiment out of two performed.
Mentions: To manipulate CD147 expression levels on HeLa cells we used the RNAi knockdown approach. HeLa-CD4 (MAGI) cells were transfected with the pSuper-retro vector (OligoEngine, Seattle, WA) containing puromycin resistance gene and siRNA corresponding to the nucleotides 470-490 of CD147 mRNA. Puromycin resistant cells displayed lower concentrations of surface CD147 than parental cell line. The cells with the highest CD147 surface expression were removed using magnetic beads followed by flow cytometric cell sorting of remaining cells. Cell clones with decreased CD147 abundance were established from the remaining cell population. Cell clones with low and medium CD147 expression were selected for further analysis in comparison to original HeLa-CD4 cell clones transduced with an empty vector (high level CD147 expressors) (Figure 1A). Importantly, the level of expression changed after several passages of cells, making it necessary to verify the expression level before each experiment.

Bottom Line: However, CD147 is not known to associate with signal transducing molecules, so other transmembrane proteins, such as proteoglycans, integrins, and CD98, were suggested as receptor or co-receptor for eCyp.Here, we manipulated CD147 expression levels on HeLa cells using RNAi and investigated the signalling and chemotactic responses to eCypA.Both Erk activation and chemotaxis correlated with the level of CD147 expression, with cells exhibiting low level expression being practically unresponsive to eCypA.

View Article: PubMed Central - HTML - PubMed

Affiliation: The George Washington University, Washington, DC 20037, USA. mtmmib@gwumc.edu.

ABSTRACT

Background: Previous studies identified CD147 as the chemotactic receptor on inflammatory leukocytes for extracellular cyclophilins (eCyp). However, CD147 is not known to associate with signal transducing molecules, so other transmembrane proteins, such as proteoglycans, integrins, and CD98, were suggested as receptor or co-receptor for eCyp. CD147 is ubiquitously expressed on many cell types, but relationship between the level of CD147 expression and cellular responses to eCyp has never been analyzed. Given the role of eCyp in pathogenesis of many diseases, it is important to know whether cellular responses to eCyp are regulated at the level of CD147 expression.

Results: Here, we manipulated CD147 expression levels on HeLa cells using RNAi and investigated the signalling and chemotactic responses to eCypA. Both Erk activation and chemotaxis correlated with the level of CD147 expression, with cells exhibiting low level expression being practically unresponsive to eCypA.

Conclusions: Our results provide the first demonstration of a chemotactic response of HeLa cells to eCypA, establish a correlation between the level of CD147 expression and the magnitude of cellular responses to eCypA, and indicate that CD147 may be a limiting factor in the receptor complex determining cyclophilin-induced Erk activation and cell migration.

No MeSH data available.


Related in: MedlinePlus