MAPping the kinase landscape of macrophage activation

Abstract

ARL11 is a tumor suppressor gene with established pro-apoptotic properties, but its function beyond this role is poorly understood. A new analysis of macrophage activation has identified ARL11 as a novel regulator of a mitogen-activated protein kinase (MAPK). These findings expand on the function of ARL11 beyond its tumor suppressor activity and highlight a novel role as a regulator of macrophage activation and inflammatory response.

Introduction

Although sequencing of the human genome provided a comprehensive map of human biology, it also uncovered the existence of vast uncharted genetic regions within our chromosomes. Further exploration of an uncharted region on chromosome 13 led to the discovery of ARL11, a small GTPase encoded by ARLTS1 (ADP-ribosylation factor-like tumor suppressor gene 1). In recent years, accumulating evidence implicates ARL11 as a tumor suppressor gene due to its ability to promote caspase-dependent apoptosis in a variety of cancer cell lines and solid tumors (1). Furthermore, it is well-established that genetic deletions and/or mutations in ARLTS1 are associated with increased familial cancer risk, and down-regulation via promoter hypermethylation is commonly observed in cancer types including breast, lung, colorectal, liver, and prostate, to name a few (2–4). Restoration of ARL11 expression, using techniques such as adenoviral transduction, has also been shown to reduce lung cancer progression (3). Despite its well-established role in cancer, ARL11 is also expressed in a variety of tissues for which its role is not yet understood.

Homologues of ARL11 are highly conserved across species, such as zebrafish, mouse, rat, Drosophila, and Arabidopsis, suggesting that it has important cellular functions (3). Mammalian ARL11 is predominantly expressed in hematologic tissues such as spleen, bone marrow, and lymph nodes (3). Given the prevalence of ARL11 expression in tissues known to play a role in the immune system, Arya et al. (5) hypothesized that further examination of immune cells may uncover a novel role for ARL11. Indeed, the authors observed that ARL11 mRNA levels were expressed in several immune cell types, predominantly in macrophages, monocytes, and neutrophils. Macrophages play a central role in inflammation and host-defense and are well-known to respond to foreign pathogens containing cell-surface lipopolysaccharide (LPS),³ an established Toll-like receptor 4 (TLR4) agonist, with a complex cascade of changes in gene expression (Fig. 1) (6, 7). Given that the intracellular pathways and outcomes of macrophage activation are well-studied and that ARL11 is highly expressed in macrophages, these cells provided an exciting point of departure to explore the role or ARL11 in inflammatory function.

Figure 1.

ARL11 modulates inflammatory response in macrophages. The binding of bacterial LPS to macrophages via TLR4 increases intracellular ARL11 expression. This promotes ARL11-mediated phosphorylation and activation of ERK1/2, thus leading to the production of pro-inflammatory mediators such as TNFα and IL-6.

To test whether ARL11 expression is altered by TLR4-mediated pathogen detection, Arya et al. (5) treated mouse macrophages with LPS and observed a concomitant increase in ARL11 protein expression after 30 min; this foundational observation supported the involvement of ARL11 in the signaling cascades of macrophage activation. To confirm the importance of this gene in macrophage activation, the authors used siRNA and short hairpin RNA to knock down ARL11 in a second mouse macrophage cell line. This led to changes in several known markers of activation, including a reduction in morphological changes, a lower capacity for Escherichia coli phagocytosis, and decreased production of pro-inflammatory markers, including the cytokines TNFα and IL-6 as well as nitric oxide.

Arya et al. (5) next investigated whether ARL11 impacted any of the canonical MAPK signaling cascades, including the ERK1/2, p38 kinase, or c-Jun N-terminal kinase (JNK1/2) pathways. The authors observed that knockdown of ARL11 in two macrophage cell lines led to a significant reduction in LPS-stimulated phosphorylation and activation of ERK1/2 and p38 kinases, but not JNK1/2. Infection with Salmonella bacteria showed a similar effect: The abundance of phosphorylated ERK1/2 and p38 were increased to a significantly greater extent in controls than in cells depleted of ARL11. Furthermore, the ability to block Salmonella replication was significantly greater in control cells. In contrast, ARL11 overexpression led to the phosphorylation and activation of ERK1/2 in an LPS-independent manner, a result that was also observed in HeLa cells despite the absence of endogenous ARL11 expression in this cell line. As a key control, the authors also observed that this result was not recapitulated with other members of the Arl family. Finally, Arya et al. (5) demonstrated that ARL11 co-localizes with ERK1/2 in a phosphorylation- and actin-dependent manner at the interior of the plasma membrane within 10 min of LPS stimulation.

Several key observations make the new results from Arya et al. (5) significant. Given that most studies utilize ARL11 ectopic/overexpression models, this report is among the first to study the function of endogenous ARL11. Indeed, it is known that prolonged ERK1/2 activation, as observed in the authors' overexpression experiments, leads to apoptosis in cancer cell lines. Thus, this study issues a call to scientists in the field to study ARL11 at endogenous concentrations to truly understand its physiological role. The study also defines new directions for future work on this intriguing protein. For example, although ARL11 and ERK1/2 could be co-immunoprecipitated, the authors indicate that a yeast two-hybrid screen did not support a direct interaction. What other factors might be involved in this complex, and how do phosphorylation and actin influence their interaction? And how does ARL11 promote ERK1/2 phosphorylation? Does ARL11 play a direct role in the p38 cascade? Additionally, this work defines a novel role of a putative tumor suppressor gene in immunological function and suggests that inhibition thereof may be used as a viable therapeutic target for the treatment of inflammatory diseases. Finally, the identification of a novel modulator of the most well-studied macrophage-activating intracellular kinases, MAPKs, demonstrates that well-established cellular pathways still harbor unexplored frontiers.