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Address correspondence to Andrei I. Ivanov, Ph.D. or Florian Rieder, M.D., Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Ave., NC22, Cleveland, OH 44195.
Address correspondence to Andrei I. Ivanov, Ph.D. or Florian Rieder, M.D., Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Ave., NC22, Cleveland, OH 44195.
Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OhioDepartment of Gastroenterology, Hepatology and Nutrition, Digestive Diseases and Surgery Institute, Cleveland Clinic Foundation, Cleveland, Ohio
Chronic inflammatory diseases cause profound alterations in tissue homeostasis, including unchecked activation of immune and nonimmune cells leading to disease complications such as aberrant tissue repair and fibrosis. Current anti-inflammatory therapies are often insufficient in preventing or reversing these complications. Remodeling of the intracellular cytoskeleton is critical for cell activation in inflamed and fibrotic tissues; however, the cytoskeleton has not been adequately explored as a therapeutic target in inflammation. Septins are GTP-binding proteins that self-assemble into higher order cytoskeletal structures. The septin cytoskeleton exhibits a number of critical cellular functions, including regulation of cell shape and polarity, cytokinesis, cell migration, vesicle trafficking, and receptor signaling. Surprisingly, little is known about the role of the septin cytoskeleton in inflammation. This article reviews emerging evidence implicating different septins in the regulation of host-pathogen interactions, immune cell functions, and tissue fibrosis. Targeting of the septin cytoskeleton as a potential future therapeutic intervention in human inflammatory and fibrotic diseases is also discussed.
Acute and chronic inflammation result in dramatic changes in tissue homeostasis. These changes include altered cellular composition and interactions between immune and nonimmune cells in affected tissues, as well as the perturbed biochemical environment driven by the release of various pro- and anti-inflammatory mediators.
Similarly, the physical properties of affected tissues change due to acute and chronic inflammation-induced development of edema, aberrant tissue remodeling, and fibrosis.
On a cellular level, inflammatory states result in functional adaptations toward accelerated recognition and elimination of the invaded pathogens, as well as enhanced production of extracellular matrix and tissue turnover.
These alterations of cellular function in inflamed tissues are mediated by reprograming of the fundamental molecular processes, including gene expression, protein synthesis, vesicle trafficking, and cytoskeletal assembly.
The cytoskeleton is a critical regulator of the architecture and function of eukaryotic cells. It comprises various filamentous structures formed via self-assembly and the polymerization of specialized proteins.
The four components of the cytoskeleton include: actin filaments, microtubules, intermediate filaments, and septin polymers. These cytoskeletal elements play crucial roles in mediating housekeeping and specialized functions in multiple cell types. Examples include regulation of cell shape and size, cell division, migration, cell–cell interactions, protein uptake and secretion, receptor signaling, etc.
Defects in the assembly and remodeling of different cytoskeletal elements play major roles in the development of various diseases, which is exemplified by tumor progression and metastasis.
The cytoskeletal regulation of tissue inflammation has also been extensively investigated. For example, the actin cytoskeleton controls the inflammatory response by regulating activation of immune cells and permeability of epithelial and endothelial barriers.
Although the roles of actin filaments, microtubules, and intermediate filament in tissue inflammation has attracted significant attention, the role of the fourth cytoskeletal element, the septin cytoskeleton, in modulating the inflammatory response remains poorly understood. This review addresses this knowledge gap by summarizing existing evidence for the involvement of the septin cytoskeleton in inflammation and tissue fibrosis and outlining possible mechanisms of such involvement.
Diversity of the Septin Protein Family
Septins (SEPT) are small GTP-binding proteins abundantly expressed in all eukaryotic organisms except for higher plants. In mammals, there are 13 septin genes (SEPT1 to SEPT12 and SEPT14) and at least 11 pseudogenes, with extensive post-transcriptional alternative splicing in many septin genes.
Existence of multiple members of the septin family (paralogs) along with expression of their differentially spliced isoforms gives rise to a complex array of different septin proteins playing either redundant or unique functional roles in different tissues. Bioinformatics analysis of the amino acid sequence homology separates mammalian septins into the following four groups named by their founding members: SEPT2 (SEPT1, -2, -4, and -5), SEPT3 (SEPT3, -9, and -12), SEPT6 (SEPT6, -8, -10, -11, and -14), and SEPT7 (SEPT7).
The groups diverge in their N-terminal extensions and C-terminal parts that contain different number of coiled-coil repeats.
Although septins are widely expressed in human tissues, some septin paralogs (SEPT2, -4, -7, -8, -9, and -10) appear to be ubiquitous, whereas others are enriched in specific tissues.
For example, high expression of SEPT3, -4, -5, and -8 is observed in the central nervous system, SEPT1, -6, and -9 are enriched in hematopoietic cells, whereas SEPT12 is especially abundant in testes.
A recent correlative analysis indicated consistent co-expression of subsets of septin paralogs in various tissues. For example, SEPT3 and SEPT5 expression displays high correlation in different brain regions, whereas a subset of septins including SEPT2, -7, -10, and to a lesser extent, SEPT11, are consistently co-expressed in non-neural tissues.
It has been suggested that septin family members with highly correlated tissue expression could be parts of the same protein complexes.
Assembly and Interactions of Septin Proteins
One of the most characteristic features of septins is their ability to self-associate, forming nonpolar rod-like hetero-oligomers and higher-order polymeric structures. Although the existence of septin rod complexes is well-documented from yeasts to mammals, their exact molecular composition is a subject of continuous debates and revisions.
Thus, recent studies have challenged a decade-old model of the organization of mammalian septin oligomers by suggesting a novel subunit arrangement in the rod complexes. According to these studies, the simplest building block of the septin cytoskeleton is composed of either six or eight protein subunits. A typical hexamer and octamer have the following subunit order: SEPT2-SEPT6-SEPT7-SEPT7-SEPT6-SEPT2 and SEPT2-SEPT6-SEPT7-SEPT9-SEPT-9-SEPT7-SEPT6-SEPT2, respectively.
Such oligomeric composition suggests that members of all four septin groups participate in the complex formation. Furthermore, according to the Kinoshita rule, members of the same septin group are interchangeable in the oligomers. For example, SEPT2 may be replaced by either SEPT4 or SEPT5, whereas SEPT9 may be replaced by either SEPT3 or SEPT12. This enables the assembly of functional septin complexes in different tissues that express distinct repertoires of septin genes. Based on this, SEPT7, the only member in its group, is predicted to be the most common and important component of the septin cytoskeleton. Indeed, several studies have demonstrated that loss of SEPT7 destabilizes other subunits of the septin oligomers and triggers their degradation.
A septin cytoskeleton-targeting small molecule, forchlorfenuron, inhibits epithelial migration via septin-independent perturbation of cellular signaling.
The septin rod oligomers spontaneously collide to form short filaments that can further assemble by end-to-end joining or lateral interactions into bundles, rings, gauzes, and sheer-like structures (Figure 1).
It is noteworthy that the composition of septin oligomers is primarily based on the in vitro reconstitution studies that utilized purified recombinant septin proteins.
The composition of such oligomers could be much more complex in vivo. Septin complexes isolated from different mammalian cells belong to either the canonical hetero-oligomers (SEPT2, -6, -7, -9; SEPT3, -5, -7; or SEPT7, -9, -11), or have unusual subunit composition inconsistent with the canonical oligomerization (SEPT2, -5, -6, -7; SEPT4, -5, -8; SEPT2, -5, etc).
It is also possible that the hetero-oligomerization is not an obligate mechanism of septin assembly and activity, and some septin family members can function as monomers or homo-oligomers. For example, loss of SEPT1, but not SEPT2, SEPT6, SEPT7, or SEPT9, has been shown to disrupt Golgi morphology in cervical cancer cells,
which selectively implicates SEPT1 in the regulation of Golgi integrity and trafficking. Furthermore, a specific SEPT4 splice isoform, SEPT4_i2, alias apoptosis-related protein in the transforming growth factor (TGF)-β signaling pathway (ARTS), was shown to selectively localize to mitochondria in kidney epithelial cells and regulate cell apoptosis.
The unique cellular activities of certain septin paralogs and splice isoforms could add another level of complexity to the septin functions and regulation in different mammalian cells and tissues.
Figure 1Organization and cellular function of the septin cytoskeleton. The assembly of the septin cytoskeleton involves initial formation of hetero-oligomers with their subsequent polymerization into higher order structures. The most well-characterized functions of septin polymers include the regulation of cell division, migration, cell–cell adhesions, and intracellular vesicle trafficking.
Importantly, a current paradigm considers the septin cytoskeleton as a universal molecular scaffold mediating the organization of other key cytoskeletal elements and their interactions with lipid membranes.
This puts the septin cytoskeleton into the center of cytoskeletal regulation. Several important molecular features of septin polymers enable such a scaffolding role. First, due to a slow rate of the GTP hydrolysis, septin filaments are more stable compared with actin filaments and microtubules.
Hence, they can serve as templates and provide a structural memory for the assembly of more labile cytoskeletal elements. Second, septins are known to physically interact with and regulate the organization of actin filaments and microtubules.
Indeed, Drosophila SEPT1/SEPT2/SEPT7 and human SEPT2/SEPT6/SEPT7 complexes, as well as human SEPT9 cross-link actin filaments into long, curved bundles.
The functional relevance of these septin-microtubule interactions was highlighted by the findings that either SEPT2, or SEPT7 depletion disrupts different microtubule populations in polarized and migrating epithelial cells.
It should be noted that the ability of septins to form multiprotein complexes is not limited to their oligomerization and interactions with actin filaments and microtubules. Published interactomes of different septin family members show a large variety of additional binding partners that include actin-binding proteins, regulators of vesicle trafficking, and signaling molecules such as kinases, phosphatases, and small GTPases.
However, functional significance of the majority of these interactions remains to be established.
Another key molecular feature of the septin oligomers is their affinity to lipid membranes. Mammalian septins preferentially bind to phosphoinositides on different cellular membranes.
Because of this, septin polymers preferentially assemble at the curvature-associated cellular structures such as cytokinetic furrows, bases of dendritic spines in neurons, branches in filamentous fungi, and intracellular lipid droplets.
Septin–membrane interactions not only promote assembly of higher-order septin cytoskeletal structures, but also significantly affect the membrane organization. For example, septin binding can induce membrane deformation, thereby controlling the shape of cellular organelles and vesicles.
Furthermore, membrane-associated septin polymers create physical barriers for the lateral diffusion of lipids and transmembrane proteins, enabling formation of distinct membrane compartments.
Finally, due to their dual affinity to membrane lipids and other cytoskeletal elements, septins physically link cellular membranes to actin filaments and microtubules, thereby creating a higher order cellular architecture.
Cellular Functions of the Septin Cytoskeleton
Given the unique ability of septins to engage in multiple interactions at different cellular compartments, it is not surprising that the septin cytoskeleton has been implicated in the regulation of a variety of cellular functions (Figure 1). Because cellular roles of septins have been described in several excellent reviews,
this review briefly summarizes their role in tissue inflammation and fibrosis. However, the canonical activity of the septin cytoskeleton in regulating cell division, also essential for septin functions in inflamed tissues has been extensively reviewed elsewhere and not discussed here.
The prominent lipid-binding ability of septins allows them to act as important regulators of intracellular vesicle trafficking. At the plasma membrane, SEPT2 participates in the formation of macropinosomes that mediate bulk uptake of the surrounding liquids.
SEPT2, SEPT6, and SEPT7 are essential for the intracellular trafficking of internalized liquid cargo by regulating its transit from early to late endosomes and delivery to lysosomes.
A different line of evidence has implicated mammalian septins in the control of internalization and intracellular fate of plasma membrane receptors, most notably members of the epidermal growth factor receptor family.
Specifically, binding with SEPT2 or SEPT9 stabilizes these receptors at the plasma membrane and attenuates their ubiquitination and degradation. Modulation of endocytosis and endosomal transit of the internalized molecules represents only one aspect of septin-dependent regulation of vesicle trafficking. In addition, septins are important for the formation of Golgi-derived vesicles and regulation of both constitutive and stimulated protein exocytosis.
The diverse septin-dependent trafficking events indicate that the septin cytoskeleton may play important roles in regulating receptor signaling on the plasma membrane, controlling transmembrane fluxes of ions and metabolites, and regulating protein secretion. The ability of septins to control multiple steps of vesicle trafficking underlies the important functions of the septin cytoskeleton in regulating host–pathogen interactions and deposition of the extracellular matrix in fibrotic tissues as discussed below.
Cell Migration and Matrix Adhesion
Another important function of the septin cytoskeleton involves regulation of cell migration. Several studies demonstrate that loss of SEPT2, SEPT7, and SEPT9 consistently attenuates wound healing in cultured epithelial and cancer cell monolayers, as well as in Xenopus embryo.
The observed promigratory activity of the septin cytoskeleton may be mediated by different mechanisms such as control of cortical microtubule assembly at the migrating cell edge,
Interestingly, SEPT2 and SEPT9 are not only essential for focal adhesion maturation, but also regulate the assembly of other adhesion structures (such as podosomes), that mediate matrix degradation.
As discussed below, septin-dependent regulation of cell migration could be an important contributor to leukocyte infiltration in the inflamed tissues.
Regulation of Cellular Signaling
In addition to interacting with other cytoskeletal structures, septins participate in additional protein–protein interactions, thereby regulating different intracellular signaling pathways. Thus, the plasma membrane–endoplasmic reticulum contact sites are important places for the septin activity in mammalian cancer cells and Drosophila neurons, where SEPT2, SEPT4, SEPT5, and SEPT7 control assembly of STIM1-ORAI1 calcium channels and regulate store-operated calcium entry.
SEPT4 has recently been identified as a novel regulator of a signal transducer and activator of transcription (STAT) 3 activity in vascular smooth muscle cells.
Septin4 prevents PDGF-BB-induced HAVSMC phenotypic transformation, proliferation and migration by promoting SIRT1-STAT3 deacetylation and dephosphorylation.
Septin4 prevents PDGF-BB-induced HAVSMC phenotypic transformation, proliferation and migration by promoting SIRT1-STAT3 deacetylation and dephosphorylation.
In contrast to STAT3 inhibition, septin binding may promote MAP kinase-dependent signaling, as supported by the direct interaction of the SEPT9_v1 isoform with c-Jun N-terminal kinase (JNK) in human mammary epithelial cells, protecting it from degradation and increasing kinase activity.
This results in a marked enhancement of JNK-dependent gene expression. Similarly, the protective effects of septin binding have significant functional consequences for hypoxia-inducible factor-1 (HIF-1) signaling. SEPT9_v1 is an important binding partner for HIF-1α in human cancer cell lines.
Targeted knockdown of SEPT9_v1 inhibits tumor growth and angiogenesis of human prostate cancer cells concomitant with disruption of hypoxia-inducible factor-1 pathway.
MSF-A interacts with hypoxia-inducible factor-1alpha and augments hypoxia-inducible factor transcriptional activation to affect tumorigenicity and angiogenesis.
This interaction protects HIF-1α protein from ubiquitination and degradation under normoxic and hypoxic conditions and markedly enhances its transcriptional activity.
MSF-A interacts with hypoxia-inducible factor-1alpha and augments hypoxia-inducible factor transcriptional activation to affect tumorigenicity and angiogenesis.
The septin-dependent regulation of STAT3, JNK, and HIF-1α signaling could be particularly important during mucosal inflammation where these signaling events are markedly up-regulated by proinflammatory cytokines and tissue hypoxia.
Septin-Dependent Regulation of Inflammation and Tissue Fibrosis
Since the discovery of the first septins in yeast cells more than 30 years ago, these proteins have been extensively studied by cell and developmental biologists. Recently, it became clear that abnormal organization and regulation of the septin cytoskeleton may contribute to the pathophysiology of different human diseases. The main focus in these studies is on understanding the roles of septin dysfunction in the development of neurodegenerative diseases and cancer.
Although several lines of evidence strongly suggest roles of septins in inflammatory disorders, very little is known about the specfics of those roles. The most plausible functions of the septin cytoskeleton during tissue inflammation are discussed below. They involve regulation of pathogen uptake, control of immune cell infiltration and activity, regulation of tissue barrier permeability, and contribution to tissue fibrosis. A summary of these functions can be found in Table 1 and Figure 2.
Table 1Functional Effects of Targeting Different Septins on Experimental Infection, Inflammation, and Fibrosis Models
Septin paralog
Interference with the septin cytoskeleton
Experimental system
Effects of the septin cytoskeleton perturbation
Reference
Host–pathogen interactions
SEPT2
siRNA-mediated knockdown
HeLa cervical epithelial cells, JEG-3 trophoblast cells
A septin cytoskeleton-targeting small molecule, forchlorfenuron, inhibits epithelial migration via septin-independent perturbation of cellular signaling.
Figure 2Mechanisms of regulation of inflammation and fibrosis through septins. This figure depicts the known or proposed impact of septins on: TGF-β and MAP kinase signaling (1); pathogen invasion, processing, and dissemination (2); exocytosis and assembly of extracellular matrix proteins (3); and immune cell migration (4). SEPT, septin; TGF-β, transforming growth factor-β.
Septin-dependent regulation of host–pathogen interactions have been initially alluded to by a study that identified SEPT9 as a major protein enriched in phagosomes responsible for the cellular entry of Listeria monocytogenes.
Subsequent studies showed crucial regulatory roles of the septin cytoskeleton during infection of cultured mammalian cells with different bacterial, fungal, or viral pathogens.
Interestingly, septins appear to have a dual role in controlling pathogen invasion and spread in the host cells. On the one hand, pathogens hijack the septin cytoskeleton to invade the host cells, as supported by septin accumulation at the pathogen entry sites during L. monocytogenes, Shigella flexneri, Salmonella typhimurium, and Candida albicans invasion.
Although mechanisms underlying septin-dependent regulation of bacterial entry remain poorly investigated, they likely involve remodeling of the cortical actin cytoskeleton. Thus, SEPT7 mediates recruitment of an important actin cytoskeletal regulator, Rho-associated kinase 2, to the S. typhimurium entry sites and promotes Salmonella-induced phosphorylation of the actin polymerizing protein, formin homology domain containing-1.
Additionally, atomic force microscopy indicates decreased viscosity and elasticity of the cell cortex following SEPT2 or SEPT11 depletion, which is consistent with altered organization of the submembranous actin cytoskeleton.
Altered organization of the cortical actin cytoskeleton in septin-deficient cells can also explain the inhibitory effects of SEPT9 depletion on the attachment of enteropathogenic Escherichia coli, which requires extensive cytoskeletal remodeling of the host cell.
Despite accelerating pathogen entry, septin cytoskeleton also acts as a negative regulator of infection by limiting survival and spread of intracellular pathogens. Septin filament assembly entraps a significant fraction of intracytosolic Shigella, including actively dividing bacteria.
More importantly, assembled septin cages recruit components of the autophagosomal machinery that accelerates the delivery of entrapped Shigella into lysosomes for degradation.
Formation of septin cages is not a unique event of Shigella infection, and also occurs during invasion of other intracellular pathogens. For example, septin filament network assembles around intracellular vacuoles containing Chlamydia trachomatis in human epithelial cells
Interestingly, encaging of Chlamydia and vaccinia virus by the host septin cytoskeleton has opposite effects on the pathogen release from the host cells. Specifically, the septin cytoskeleton prevents vaccinia virus release and cell-to-cell dissemination while promoting Chlamydia extrusion.
Finally, a different mechanism exploiting the lipid-binding ability of the septin cytoskeleton was shown to drive hepatitis C virus (HCV) infection of liver cells.
HCV infection markedly accelerates SEPT9 expression and septin filament assembly. This results in accumulation of intracellular lipid droplets, which is dependent on septin filament interactions with phosphoinositide and microtubules. Such septin-driven formation of lipid droplets is crucial for efficient HCV replication.
While a growing number of studies focus on the septin-dependent regulation of pathogen interactions with cultured cells in vitro, almost nothing is known about the ability of the septin cytoskeleton to regulate pathogen infection in vivo. The only study addressing this reported that septins restrict Shigella infection and the associated inflammatory response in zebrafish larvae.
In this study, loss of SEPT7 increased bacterial burden, which was linked to overactivation of IL-1β signaling and increase in bacteria-induced neutrophil death.
Interestingly, pharmacologic inhibition of the inflammatory response using IL-1 receptor antagonist rescued neutrophil survival and decreased bacterial burden in septin-deficient zebrafish. Together, these studies strongly suggest that the septin cytoskeleton could serve as an important regulator of host–pathogen interactions and inflammatory responses triggered by bacterial or viral infections.
Immune Cell Development and Functions
Different septin paralogs are abundantly expressed in human macrophages, lymphocytes, and dendritic cells.
However, the role of the septin cytoskeleton in regulation of immune cell development and functions remains poorly understood (Table 1). In macrophages, SEPT2 and SEPT11 accumulate at the phagosomes in actin cytoskeleton-dependent fashion, and SEPT2 depletion was shown to inhibit Fc receptor–mediated phagocytosis.
Disruption of the septin cytoskeleton by SEPT7 depletion causes profound alterations in T lymphocyte cell morphology manifested by the excessive membrane blebbing and increased uropod length.
The disruption of the septin cytoskeleton also has a complex effect on T-cell motility such that SEPT7-deficient T cells exhibit decreased crawling velocity during random planar migration, but are able to more efficiently transmigrate through confined spaces, such as the small pores in the Transwell membrane filters.
It has been suggested that decreased rigidity of the cell cortex in septin-deficient T cells increases the ability of the cell membrane to form protrusions, which impairs the processive and directional movement of the cell in open spaces. By contrast, such cortical destabilization allows for higher deformability of septin-depleted cells that accelerates their passing through narrow spaces.
Disruption of the septin cytoskeleton negatively impacts other aspects of T-cell development and functions. Thus, transgenic mice with T-cell–specific ablation of SEPT9 are characterized by a decreased number of mature peripheral T cells, especially the CD8+ T-cell population.
These maturation defects are associated with the decreased proliferation capacity of SEPT9-deficient CD8+ T cells. Similarly, conditional knockout of SEPT7 in T lymphocytes results in the diminished CD8+ cell population in aged mice.
Additionally, isolated SEPT7-deficient CD8+ T cells show markedly reduced proliferation and cytokinesis failure after stimulation with soluble immune activators such as cytokines, phorbol ester, or ionomycin.
In addition to playing important roles in regulating pathogen engagement and immune cell responses, septin cytoskeleton serves as essential regulator of the integrity and permeability of tissue barriers (Table 1). Indeed, disruption of epithelial and vascular endothelial barriers is a common feature of different inflammatory processes.
Limited and transient increased barrier permeability has beneficial effects by allowing tissue influx of immune cells and efficient pathogen clearance. However, poorly controlled and prolonged disruption of epithelial and endothelial barriers is detrimental because it increases body exposure to environmental pathogens and stressors, and exaggerates the inflammatory response.
Permeability of epithelial and endothelial barriers is regulated by specialized adhesive structures, adherens and tight junctions, which are regulated by the underlying cortical actomyosin cytoskeleton.
Emerging evidence suggests that the septin cytoskeleton acts as an important regulator of epithelial and endothelial junctions. For example, SEPT2 is enriched at intercellular junctions in cultured human endothelial
A septin cytoskeleton-targeting small molecule, forchlorfenuron, inhibits epithelial migration via septin-independent perturbation of cellular signaling.
These examples demonstrate that septin-dependent stabilization of epithelial and endothelial barriers could serve as a protective mechanism limiting immune cell exposure to environmental pathogens during tissue inflammation.
Tissue Fibrosis
Limited evidence, primarily derived from the liver and kidney, implicates the septin cytoskeleton in the regulation of aberrant tissue repair and fibrosis (Table 1). The only comprehensive characterization of septin expression in fibrotic tissues was performed in a mouse model of kidney fibrosis.
A gene expression analysis for different septins (SEPT1 and SEPT4 through SEPT11) demonstrates a significant up-regulation of all septin transcripts in the fibrotic kidney compared with controls. Immunohistologic experiments detect SEPT7 and SEPT8 protein up-regulation primarily in the tubulointerstitial region of the fibrotic kidney. Myofibroblasts are considered the chief profibrotic cell type across organs. Interestingly, septins were shown to significantly colocalize with the myofibroblast marker α-smooth muscle actin in the diseased kidneys.
SEPT4 and SEPT6 were also found up-regulated in different rodent models of liver fibrosis, where they were associated with hepatic stellate cells (HSC).
However, given a recent report that shows down-regulation of SEPT9 expression in carbon tetrachloride–induced liver fibrosis, septin expression could be a subject of more complex regulation in fibrotic tissues.
Functional effects of selected septins on fibrogenesis were examined in a limited number of studies that utilized knockout mice models and adenovirus-mediated manipulation of septin expression in vivo. Mice with total knockout of SEPT4 display exaggerated hepatic fibrosis in three different experimental models (carbon tetrachloride, and cholestasis- and steatohepatitis-induced fibrosis).
These profibrotic effects of SEPT4 deletion are linked to the increased activation and collagen-producing ability of HSC, the major myofibroblast precursor cell type in the liver. Supporting this loss of function data, adenoviral-mediated overexpression of SEPT4 suppresses hepatic fibrosis induced by Schistosoma japonicum infection in mice.
Antifibrotic effects of SEPT4 overexpression are associated with inhibition of HSC activity and their increased apoptosis. It is noteworthy that SEPT4 was shown to be a potent regulator of spontaneous and TGF-β–induced apoptosis in different tissues.
Indeed, shRNA-mediated depletion of SEPT6 in vivo attenuates thioacetamide-induced hepatic fibrosis in rats by inhibiting HSC activation and decreasing the excessive extracellular matrix deposition seen in fibrosis.
Finally, SEPT8-null mice do not show any differences in the development of kidney fibrosis as compared with wild-type controls, thereby suggesting dispensability of SEPT8 in this disease model.
At present, it is unclear whether the conflicting evidence reflects peculiar roles of different members of the septin family in fibrosis, or tissue-specific effects of the septin cytoskeleton on fibrogenesis. In addition, a temporal role of the septin cytoskeleton in early versus late tissue damage has not been evaluated yet.
On a cellular level, septin-dependent regulation of tissue fibrosis is likely linked to the central profibrotic signaling pathway involving TGF-β. TGF-β is known to modulate septin expression and localization by up-regulating SEPT6
Septins themselves could act as positive regulators of TGF-β signaling by stimulating expression of this growth factor and promoting TGF-β–driven Smad signaling, as was shown for SEPT6 and SEPT9 actions in HSC
respectively. These data indicate that the septin cytoskeleton could be part of an important fibrogenic positive feedback loop mechanism involving TGF-β–induced up-regulation of septins, which in turn triggers further increase in TGF-β level and activity. Septins modulate other signaling pathways important for fibrogenesis in HSC. For example, SEPT6 up-regulates the activity of all three major MAP kinases: ERK, JNK, and p38,
The Septin Cytoskeleton as a Potential Target for Therapeutic Interventions
Emerging functions of the septin cytoskeleton in regulating various aspects of the immune response and tissue remodeling during inflammation and fibrosis raise the important question of whether septins could be successfully targeted to develop novel anti-inflammatory and antifibrotic therapies. In general, functional activity of different cytoskeletal structures such as actin filaments and microtubules could be modulated by cell-permeable small molecules that cause either stabilization or disassembly of targeted cytoskeletal structures. The feasibility of clinical application of such compounds is illustrated by the successful use of microtubule-stabilizing taxane class drugs as anticancer agents. However, pharmacologic modulation of the septin cytoskeleton, while possible, remains at the very early stage of exploration. The experimental toolbox of septin-modulating compounds is very narrow with only one small molecule, forchlorfenuron (FCF), being extensively used to target the septin cytoskeleton in different experimental systems.
Interestingly, such FCF-induced stabilization of the septin cytoskeleton mimics functional effects of the depletion of key septin paralogs by inhibiting cell division, migration, vesicle trafficking, calcium entry, and cell–cell adhesions.
Despite diverse effects on cell function, FCF itself may not be suitable for clinical use in its current form. It has a relatively low affinity for septin filaments and hence is only active at high, submillimolar concentrations. Furthermore, possible off-target effects and animal tissue toxicity of FCF have been reported.
A septin cytoskeleton-targeting small molecule, forchlorfenuron, inhibits epithelial migration via septin-independent perturbation of cellular signaling.
Nevertheless, studies utilizing FCF have laid the ground for the development of next-generation septin inhibitors. We expect that these inhibitors will be first developed as anticancer drugs, given the substantial body of evidence about mutations or altered expression of different septin paralogs being associated with tumorigenesis.
Not much is known about the possibility of pharmacologic inhibition of the septin cytoskeleton during inflammation and tissue fibrosis. Further studies are required to examine potential utilization of FCF and its derivatives for the development of novel anti-inflammatory and antifibrotic therapies.
Conclusion
The septin cytoskeleton represents a critical component of cellular architecture that is known to mediate a variety of housekeeping and specialized functions. Most known for their roles in normal tissue development and homeostasis, septins are increasingly recognized as important contributors to human diseases. A significant body of literature links the altered expression and mutations of different septins with the development of neurological diseases and cancer. Much less is known about the role of the septin cytoskeleton in regulating tissue inflammation and fibrosis. As demonstrated in this review article, exciting recent developments in this field include discovering the roles of septins in the regulation of host interactions with bacterial and viral pathogens, modulation of lymphocytes differentiation, and profibrotic signaling (Table 1 and Figure 2). However, a number of important questions remain to be answered. For example, it is crucial to understand whether, as part of the same cytockeletal structure, different septins similarly regulate inflammatory and fibrotic responses, or whether members of the septin protein family have unique functional roles. It would be important to examine how septins regulate signaling by pattern recognition receptors that sense different pathogens, as well as signaling by different inflammatory cytokines and growth factors. Given the known roles of septins in the regulation of vesicle trafficking and the Golgi, it should be a research priority to determine whether the septin cytoskeleton controls secretion of different inflammatory factors by activated immune cells and regulates deposition of the extracellular matrix in fibrotic tissues. There is obvious need for extensive in vivo studies involving mouse models with tissue-specific knockout of different septins. From a therapeutic standpoint, it is critical to understand whether the septin cytoskeleton could be a druggable target during inflammation and tissue fibrosis, and to develop specific pharmacologic modulators of septin cytoskeletal assembly and dynamics. We are still at the very beginning of the exploration in this exciting and potentially fruitful research field, and future studies will pave the way for the modulation of septins to treat human inflammatory and fibrotic diseases.
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A septin cytoskeleton-targeting small molecule, forchlorfenuron, inhibits epithelial migration via septin-independent perturbation of cellular signaling.
Septin4 prevents PDGF-BB-induced HAVSMC phenotypic transformation, proliferation and migration by promoting SIRT1-STAT3 deacetylation and dephosphorylation.
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Supported by NIH- NIDDK grants RO1 DK108278 (A.I.I.), K08 DK110415 , and R01 DK123233 (F.R.); and the Kenneth Rainin Foundation Synergy Award (A.I.I. and F.R.).
Disclosures: F.R. is a consultant or on an advisory board for Agomab, Allergan, AbbVie, BMS, Boehringer Ingelheim, Celgene, CDISC, Cowen, Falk Pharma, Genentech, Gilead, Gossamer, Guidepoint, Helmsley, Index Pharma, Janssen, Koutif, Metacrine, Morphic, Pfizer, Pliant, Prometheus Biosciences, Receptos, RedX, Roche, Samsung, Takeda, Techlab, Theravance, Thetis, and UCB.
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