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Role of microtubules in LPS-induced macrophage inflammatory protein-2 production from rat pneumocytes
N Isowa1, S H Keshavjee, M Liu
1Thoracic Surgery Research Laboratory, Toronto General Hospital, University Health Network, Toronto, Ontario, Canada M5G 2C4.
Abstract:
We have recently demonstrated that primary cultured rat pneumocytes produce macrophage inflammatory protein-2 (MIP-2) in response to lipopolysaccharide (LPS) stimulation. In this study, we found that brefeldin A, by blocking anterograde transport from the endoplasmic reticulum (ER) to the Golgi apparatus, decreased LPS-induced MIP-2 in the culture medium and increased its storage in cells. This suggests that MIP-2 is secreted via a pathway from the ER to the Golgi apparatus, a process commonly regulated by microtubules. We further found that LPS induced depolymerization of microtubules as early as 1 min after LPS stimulation, and it lasted at least for 4 h. Preventing depolymerization of microtubules with paclitaxel (Taxol; 10 nM to 10 microM) partially inhibited LPS-induced MIP-2 production, whereas the microtubule-depolymerizing agents colchicine (1-10 microM) and nocodazole (1-100 microM) increased LPS-induced MIP-2 protein production without affecting MIP-2 mRNA expression. These results suggest that in pneumocytes, LPS-induced microtubule depolymerization is involved in LPS-induced MIP-2 production and that secretion of MIP-2 from pneumocytes is via the ER-Golgi pathway.
Insights
Lipopolysaccharide (LPS) stimulates rat pneumocytes to produce macrophage inflammatory protein-2 (MIP-2). Microtubule depolymerization is crucial for LPS-induced MIP-2 production and secretion via the ER-Golgi pathway.
Area of Science:
- Cell Biology
- Immunology
- Respiratory Medicine
Background:
- Primary cultured rat pneumocytes produce macrophage inflammatory protein-2 (MIP-2) upon lipopolysaccharide (LPS) stimulation.
- The secretion pathway and regulation of MIP-2 in pneumocytes remain incompletely understood.
Purpose of the Study:
- To investigate the role of intracellular transport and microtubules in LPS-induced MIP-2 production and secretion by rat pneumocytes.
- To elucidate the mechanism by which LPS affects MIP-2 secretion.
Main Methods:
- Treatment of rat pneumocytes with LPS, brefeldin A, paclitaxel, colchicine, and nocodazole.
- Measurement of MIP-2 levels in cell culture medium and within cells.
- Assessment of microtubule integrity and MIP-2 mRNA expression.
Main Results:
- Brefeldin A blocked MIP-2 secretion, suggesting ER-Golgi pathway involvement.
- LPS induced rapid and sustained microtubule depolymerization.
- Paclitaxel partially inhibited LPS-induced MIP-2 production, while colchicine and nocodazole enhanced it without affecting mRNA levels.
Conclusions:
- MIP-2 secretion from pneumocytes occurs via the endoplasmic reticulum (ER)-Golgi pathway.
- LPS-induced microtubule depolymerization is integral to the regulation of MIP-2 production in pneumocytes.
- Microtubule dynamics play a critical role in modulating inflammatory mediator secretion.