Monocyte differentiation to macrophages leads to HIF 1a transloca

Monocyte differentiation to macrophages leads to HIF 1a translocation We considered whether differentiation of human mono cytes into macrophages using hM CSF also caused hypoxia induced translocation of HIF 1a into the nucleus. The macrophage nuclear sellckchem fraction was identified using the location of Lamin B, the location of actin was not used as this may be found in the nuclear fraction of macrophages due to alterations in the cytoskeleton of macrophages after stimulation, as described by Hartwig and Janmey. Incubation of monocytes with 25 nM hM CSF over 7 days led to differentiation into hMDM. Inhibitors,Modulators,Libraries After incubation Inhibitors,Modulators,Libraries of monocytes and hMDMs for 5 h under hypoxia, HIF 1a in the monocytes could only be detected in the cytosolic compartment, while in hMDMs HIF 1a was seen to reside in the nuclear extract.

We also Inhibitors,Modulators,Libraries considered whether any possible prior adhesion of human monocytes to endothelial cells, as an initial step of migration, could initiate the translocation of HIF 1a into the nucleus. Human monocytes were incubated for 5 h in plates with wells coated with human microvascular endothelial cells, under hypoxia and under nor moxia. The cellular localization of HIF 1a was investi gated. We observed that co culture with endothelial cells of this type did not induce accumulation of HIF 1a in the nucleus of the monocytes. HIF 1a was found exclusively in the cytosol fraction of monocytes, as assessed by immu noblot, HIF 1a was not detectable under nor moxia. Taken together, these findings suggest that adhesion of monocyte to the vascular wall is not sufficient for translocation of HIF 1a Inhibitors,Modulators,Libraries to the nucleus.

Hypoxia induced gene expression Inhibitors,Modulators,Libraries of human monocytes versus hMDMs Next we compared the expression of selected hypoxia induced genes in human monocytes and hMDMs. We examined genes that have been identified as typical HIF 1 target genes such as the glycolysis enzymes LDHA and PGK1, and the chemokine receptor CXCR4. In monocytes incubated under hypoxic conditions in contrast to normoxia genes for LDHA and CXCR4 were significantly upregulated, although HIF 1a is not present in the nucleus, the gene for PGK1 showed increased expression but this did not reach sta tistical significance. HIF1A as a target gene of HIF 1 itself is regulated at the protein level and therefore showed no measurable induction. Macrophages under normoxia showed higher expression of the genes LDHA and PGK1 than monocytes.

There was no significant difference in the expression of these genes under hypoxia versus normoxia, presumably because metabo lism had already been switched from oxidative phos phorylation respiration to anaerobic glycolysis during differentiation. inhibitor purchase Similar findings were observed for CXCR4, where hypoxic incubation did not lead to a significant increase of CXCR4 expression in hMDMs. Also in hMDMs, hypoxia did not induce any measurable upre gulation of HIF1A.

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