Caveolin-1 Knockdown by Small Interfering RNA Suppresses Responses to the Chemokine Monocyte Chemoattractant Protein-1 by Human Astrocytes

Abstract

Astrocytes regulate the integrity of the blood-brain barrier and influence inflammatory processes in the central nervous system. The pro-inflammatory chemokine monocyte chemoattractant protein-1 (MCP-1), which is both released by and stimulates astrocytes, is thought to play a crucial role in both these activities. Because astrocytes have been shown to possess caveolae, vesicular structures that participate in intracellular transport and signal transduction events, we reasoned that expression of the major structural protein of these organelles, caveolin-1, might feature critically in the cellular responses to MCP-1. To test this hypothesis, caveolin-1 level was "knocked down" in human astrocyte cultures by using a small interfering RNA approach. This method resulted in efficient (>90% loss) and specific knockdown of caveolin-1 expression while sparring glial fibrillary acidic protein as well as several other proteins involved in endocytosis. Astrocytes suffering caveolin-1 loss showed diminished ability to down-modulate and internalize the MCP-1 receptor (CCR2) in response to exposure to this chemokine and also demonstrated significantly reduced capacity to undergo chemotaxis and calcium flux when MCP-1-stimulated. The results highlight a potentially prominent role for caveolae and/or caveolin-1 in mediating astrocyte responses to MCP-1, a feature that might significantly dictate the progression of inflammatory events at the blood-brain barrier.

Chemokine receptor internalization and intracellular trafficking

“…Strong affinity interactions result in slow recycling or degradation, while low affinity interactions may result in rapid recycling [100][101][102]. Interestingly, studies on the chemotactic protease-activated receptor-2 (PAR-2) demonstrate that the receptor utilizes a β-arrestin-dependent mechanism to sequester activated ERK1/2 in the pseudopodia [47]. This raises the possibility that intracellular trafficking of chemokine receptors mediated by β-arrestin may have important implications for localization of signals.…”

“…However, non-specific effects of cholesterol depletion cannot be ruled out. In addition, studies conducted in astrocytes reveal that the inhibition of CCL2-triggered down-modulation and internalization of CCR2 by knocking down caveolin-1, a key component for caveolae, significantly inhibits chemotaxis and calcium mobilization [47].…”

“…The ability of RNA-interference to knockdown endogenous caveolin-1 is a useful technique to assess caveolin-dependent internalization pathways. Astrocytes exhibiting caveolin-1 knockdown showed diminished ability to down-modulate and internalize CCR2 in response to ligand stimulation [47]. Moreover, there may be cell type differences in utilization of lipid rafts for chemokine receptor internalization.…”

Distinct modes of molecular regulation of CCL3 induced calcium flux in monocytic cells

“…We have previously shown that CCR5 internalisation can be inhibited by treatment with sucrose, nystatin and filipin, which inhibit clathrin-coated pit internalisation and affect caveolae in the cell membrane, respectively [2]. There have been reports about a number of receptors, like CCR4 [3] or CCR2 [4] where caveolae are important for receptor internalisation. Furthermore it has been reported that chemokines bind preferentially to receptors which are associated with cholesterol and sphingolipid-rich 3 lipid raft microdomains in the plasma membrane [5].…”

The Ras-MAPK pathway downregulates Caveolin-1 in rodent fibroblast but not in human fibroblasts: implications in the resistance to oncogene-mediated transformation

“…Finally, in order to evaluate the effect of Caveolin-1 downregulation on the oncogenic transformation of normal HDFs, TIG3/TSR and BJ/TSR cells were transduced with a retroviral vector expressing a short interfering dsRNA for caveolin-1 (shCav422) (Ge and Pachter, 2004). These transduced cells were then selected with zeocine, resulting in a bulk population of (Figure 3a).…”