Protein Kinase C β Is Required for Human Monocyte Chemotaxis to MCP-1

Abstract

Monocyte chemoattractant protein 1 (MCP-1) is important in attracting monocytes to sites of inflammation. Using predominantly pharmacological approaches, prior studies have indicated that serine/ threonine kinases are involved in the MCP-1-induced signaling pathways. We report here that there is substantial inhibition of MCP-1-stimulated chemotaxis of human monocytes treated with inhibitors selective for the subset of serine/threonine kinases, protein kinase C (PKC). Selective inhibitors of PKC such as GF109203X and Calphostin C both caused ϳ80% inhibition of chemotaxis. Because these pharmacological inhibitors do not specifically inhibit individual PKC isoforms, we chose to use antisense oligodeoxyribonucleotides (ODN) to specifically reduce PKC isoform expression, first by inhibiting expression of the conventional PKC family, and next by using specific antisense ODN for PKC␣ and PKC␤. Conventional PKC-antisense ODN treatment completely and significantly inhibited monocyte chemotaxis to MCP-1, whereas sense-control ODN caused no significant inhibition. PKC␤-antisense ODN caused 89.2% inhibition of chemotaxis at its highest dose. In contrast, PKC␤-sense ODN and PKC␣-antisense and -sense ODN were without effect. Further studies evaluating the calcium response that is triggered upon MCP-1 interaction with its receptor, CCR 2 , indicate that this response is not altered by antisense or sense ODN treatment, thus supporting our hypothesis that PKC␤ is critical for post-receptor signal transduction downstream of the immediate calcium signal. These data contribute to our developing understanding of the signal transduction pathways involved in the chemotactic response of human monocytes to MCP-1 and uniquely identify the requirement for the PKC␤ isoform in this important process.

In vivo validation of signaling pathways regulating human monocyte chemotaxis

“…Interestingly, a significant reduction was also observed in the basal migration of GFP-PKCβ-DN expressing monocytes in the absence of MCP-1. These data support our previous observations, using pharmacologic inhibitors and antisense oligonucleotides, that identified an important regulatory role of PKCβ in monocyte chemotaxis to MCP-1 in vitro (Carnevale and Cathcart, 2003). They also highlight the success of our transfection procedure and feasibility of using GFP-expressing monocytes for in vivo studies.…”

“…Previous studies from our lab identified PKCβ as a critical regulatorof human monocyte chemotaxis to MCP-1 in vitro using pharmacologic inhibitors and specific antisense oligonucleotides (Carnevale and Cathcart, 2003). To functionally validate our method of transfection, we investigated the role of PKCβ in monocyte migration by overexpressing GFP-PKCβ-WT as well as the dominant negative mutant, GFP-PKCβ-DN plasmid DNA.…”

“…Our studies to date have identified several unique signaling pathways that regulate monocyte chemotaxis in vitro (Carnevale and Cathcart, 2001;Carnevale and Cathcart, 2003) but it is important to validate the role of these pathways in vivo in relevant inflammatory responses. One kinase shown to regulate the in vitro chemotactic response of human monocytes to MCP-1 is PKCβ (Carnevale and Cathcart, 2003).…”

“…One kinase shown to regulate the in vitro chemotactic response of human monocytes to MCP-1 is PKCβ (Carnevale and Cathcart, 2003). To confirm the relevant in vivo role for PKCβ, we optimized transfection conditions in primary human monocytes and then transfected them with wild type and dominant negative PKCβ cDNA.…”

“…Since transfection of primary monocytes with cDNA was not available until recently, we have instead used antisense oligonucleotides as specific inhibitors of mRNA and protein expression to explore monocyte signaling (Li and Cathcart, 1994;Li and Cathcart, 1997;Roy and Cathcart, 1998;Li et al, 1999;Bey and Cathcart, 2000;Carnevale and Cathcart, 2001;Bey and Cathcart, 2002;Zhao et al, 2002;Carnevale and Cathcart, 2003;Bey et al, 2004;Xu et al, 2004;Zhao et al, 2005;Bhattacharjee et al, 2006;Li et al, 2007). We report here the optimization of an efficient method for transfecting primary monocytes that has dramatically expanded the conventional approaches for studying potential signaling pathways involved in the pathogenic processes accompanying several inflammatory diseases including atherosclerosis.…”