Differential regulation of Rab GTPase expression in monocyte-derived dendritic cells upon lipopolysaccharide activation: a correlation to maturation-dependent functional properties

Abstract

The regulation of Rab expression to modulate cellular function has recently been proposed. Dendritic cells are a prototypic example of cells that drastically alter their function in response to environmental cues by reducing endocytosis, secreting cytokines, changing surface protein repertoires and altering morphology and migration. This is not a binary event, but is subject to fluctuations through the activation process, termed maturation. Consequently, DCs transiently increase endocytosis and production of major histocompatibility complex class II molecules, and secrete inflammatory cytokines in infected tissues before migrating to secondary lymph nodes and releasing T cell polarizing factors. All these cellular processes rely on intracellular membrane transport, which is regulated by Rab family GTPases and their diverse effectors. Here we examine how the Rabs likely to be involved in these functions are regulated throughout DC maturation. We find that Rab expression is altered upon lipopolysaccharide-induced activation, and discuss how this correlates to the reported functions of these cells during maturation.

Figure 1. Rabs show large variation in expression levels in iDCs.

Quantitative RT-PCR shows expression levels of the targeted Rab GTPases prior to activation of moDCs. Means and SD are shown on a logarithmic scale, n=6. Expression levels have been normalized to the reference gene GADPH.

Figure 2. Rabs involved in endocytosis exhibit an early increase after LPS activation.

Quantitative RT-PCR shows expression levels of Rab5a (A), Rab8a (B), Rab35 (C), Arf6 (D) and Rab21 (E) in moDCs, after LPS stimulation for the indicated time periods. All data are normalized to initial iDC expression levels. Means and SD are shown, n = 6, * P<0.05.

Figure 3. Endosomal recycling Rabs increase in the early phase of DC maturation.

Quantitative RT-PCR shows expression levels of the targeted Rab GTPases in moDCs, after LPS stimulation for the indicated time periods. All data are normalized to initial iDC expression levels. Rab4b (A) and Rab11a (B) are involved in short and long loop recycling, respectively. Means and SD are shown, n = 6.

Figure 4. Late endocytic Rabs are differently expressed during DCs maturation.

Quantitative RT-PCR shows expression levels of the targeted Rab GTPases in moDCs, after LPS stimulation for the indicated time periods. All data are normalized to initial iDC expression level. Rab7a (A) and Rab9 (B) show weak responses to DC activation compared to Rab7b (C). Means and SD are shown, n = 6, *P<0.05.

Figure 5. Rabs involved in delaying phagosomal maturation are differentially regulated during the maturation process.

Quantitative RT-PCR shows expression levels of Rab14 (A), Rab22a (B) and Rab27a (C) in moDCs, after LPS stimulation for the indicated time periods. All data are normalized to initial iDC expression level. Means and SD are shown, n = 6. *P<0.05, **P<0.01.

Figure 6. Rab GTPases involved in exocytosis

alter their expression as the DC matures. Quantitative RT-PCR shows expression levels of Rab27b (A), Rab6a (B), Rab10 (C) and Rab3b (D) in moDCs, after LPS stimulation for the indicated time periods. All data are normalized to initial iDC expression levels. Means and SD are shown, n = 6, *P<0.05.

Figure 7. Protein levels of GTPases during DC maturation.

(A) Protein lysate from moDCs stimulated with LPS for the indicated time points were run on SDS-PAGE and subjected to Western blot analysis using anti-Rab7b, anti-Rab27a, anti-Rab21, anti-Arf6, anti-Rab9 and anti-tubulin antibodies. (B) The intensity of the bands was quantified by densitometry, and normalized against tubulin. Protein levels are relative to the initial iDC levels. Mean and SD are shown, n= 3.