antly, the e pression of CCR2, but not CCR1, was selectively downregulated, suggesting that the loss of this chemokine receptor is a consequence of monocyte etc differentiation. This downregu lation was observed at the level of cell surface receptor e pression, mRNA e pression, and transcription. Clearly, these are specific regulatory events since the levels of CCR1 mRNA are not affected by either combination of pharmacologic agents. However, when THP 1 cells were treated with PMA or PMA plus ionomycin in the presence of stau rosporine, differential results were obtained PMA medi ated modulation of CCR2 was sensitive to the inhibitory effects of staurosporine, whereas staurosporine concentrations as high as 200 nM failed to block PMA plus ionomycin induced downregulation of CCR2.

Stau rosporine alone did not promote the loss of either CCR2 or CCR1. These results indicate that staurosporine defines a dichotomy in the regulation of CCR2 e pression by PMA versus PMA plus ionomycin that had not previously been appreciated. Staurosporine, itself, is a broad spectrum inhibitor of pro tein kinases including PKA, PKC, and PKG. PMA has clas sically been shown to act almost e clusively through PKC and this would e plain why staurosporine was able to block the PMA induced downregulation of CCR2. By inference, PMA plus ionomycin would appear to act through a signal transduction pathway that is not inhib ited by staurosporine and presumably this means that sec ond messengers other than PKA, PKC and PKG are involved. To that end, calcineurin, a calcium sensitive phosphatase may be a target for PMA plus ionomycin.

An increase in the intracellular calcium concentra tion promotes a conformational change in calcineurin, which then dephosphorylates and activates the transcription fac tor NFAT facilitating its translocation to the nucleus. In addition, it has been shown that PMA enhances the cal cium sensitivity Brefeldin_A of NFAT, thus creating a synergistic signal. This synergy may result from de novo synthesis and post translational modification of another transcrip tion factor termed activating protein 1, AP 1. Indeed, NFAT proteins show a characteristic ability to co operate with AP 1 in DNA binding and transactivation. Interestingly, in the region of the CCR2 promoter that we cloned there are two putative binding sites for AP 1 TCA and three putative binding sites for NFAT as determined by the MatInspecter transcription factor bind ing site analysis program.

It has also been suggested that additional transcription factors including OCT1 and C EBP can act synergistically with NFAT www.selleckchem.com/products/mek162.html and again there are multiple binding sites for each of these DNA binding pro teins in the CCR2 promoter, although at this stage we have no evidence to suggest that they are involved in the physiological regulation of CCR2 gene e pression. A requirement for co operation and cross talk between these two pharmacologic agents is further supported by the fact that ionomycin alone was unable to down modul