Insights into the role of progranulin in immunity, infection, and inflammation

Review on the role of progranulin in immunity, infection, and inflammation, and its therapeutic potential in treating inflammatory conditions.

Abstract

PGRN, a pleiotrophic growth factor, is known to play an important role in the maintenance and regulation of the homeostatic dynamics of normal tissue development, proliferation, regeneration, and the host-defense response and therefore, has been widely studied in the fields of infectious diseases, wound healing, tumorigenesis, and neuroproliferative and degenerative diseases. PGRN has also emerged as a multifaceted immune-regulatory molecule through regulating the signaling pathways known to be critical for immunology, especially TNF/TNFR signaling. In this review, we start with updates about the interplays of PGRN with ECM proteins, proteolytic enzymes, inflammatory cytokines, and cell-surface receptors, as well as various pathophysiological processes involved. We then review the data supporting an emerging role of PGRN in the fields of the “Cubic of I”, namely, immunity, infection, and inflammation, with special focus on its regulation of autoimmune syndromes. We conclude with insights into the immunomodulating, anti-inflammatory, therapeutic potential of PGRN in treating diseases with an inflammatory etiology in a vast range of medical specialties.

Introduction

The cubic of I, or I³, is a term used to describe three closely related and interdependent physiological and pathological processes in the organism, which include immunity, infection, and inflammation. Immunity's functions encompass the protection of the body from attack by pathogenic micro-organisms, surveillance over mutated somatic cells, and regulation of the appropriate strength and intensity of the immune system's reactions to antigenic stimuli. Infection is the process by which pathogens interfere with host-defense responses and cause pathology-generating morbidities. Inflammation takes place as a result of the battle between immunity and infection and also occurs in other pathological conditions, including wound-healing [1] and autoimmune disorders. A vast array of cell types and cytokines is involved in these processes. For instance, macrophages and neutrophils are the major cells recruited in the first line of host defense [2]. They kill microbial pathogens directly and present antigens to trigger adaptive immunity, along with releasing cytokines to cause inflammation. T cells and B cells are involved in the second line of host defense [3]. They recognize antigens meticulously through specific TCRs or BCRs.

Many cytokines are released in the competition between immunity and infection, as well as during inflammation. The network-like collaboration between cytokines is essential to fine-tune the immune response and its reactive inflammatory state. However, dysregulated cytokine production causes the development of autoimmune disorders or simply, the persistence of infection, in turn, maintaining a chronic pathological inflammatory state [4]. Among the numerous cytokines studied, TNF-α has been noted to be especially important [5], as it is at the top of the hierarchy, upstream of the cascade of inflammation, and likewise, has multiple complex functions in the immune system. TNF-α signaling blockers have been applied successfully in clinical trials to treat a wide range of inflammatory diseases, including RA (reviewed in refs. [6, 7]) and IBDs (reviewed in refs. [8, 9]).

PGRN, also known as GRN–epithelin precursor [10], proepithelin [11, 12], acrogranin [13], and GP88/PC cell-derived growth factor [14], is a 593-aa growth factor that is secreted from cells mediated by its signal peptide. PGRN contains 7½ repeats of a cysteine-rich motif (CX5–6CX5CCX8CCX6CCXDX2HCCPX4CX5–6C) in the order, P–G–F–B–A–C–D–E, where A–G are full repeats, and P is the one-half motif [15]. PGRN is digested into 6-kDa GRN peptides by many proteinases, including matrix metalloproteinase 9, 12, and 14, elastase, and proteinase 3 [16–20]. There are many distinctive and contrasting functions executed by PGRN and GRN. For instance, PGRN, as opposed to GRN, blocks the TNF-α-induced respiratory burst in neutrophils [17], whereas GRN B, but not PGRN, stimulates IL-8 expression in epithelial cells [17]. It is not clear, however, by which mechanism the single 6-kDa GRNs mediate its biological function, as most PGRN-binding membrane receptors need more than one GRN domain (see Table 1).

Table 1. PGRN-Interacting Proteins.

PGRN-associated proteins	Interaction sites	Interaction domain PGRN	Function	References
ADAMTS7	Matrix	GRN A–G	Inhibition of ADAMTS 7 activity	[45]
ADAMTS12	Matrix	GRN A–G	Inhibition of ASAMTS 12 activity	[45]
COMP	Matrix	GRN A	COMP enhances PGRN-induced proliferation	[40]
Perlecan domain V	Matrix	GRN F + B	Promotion of tumor growth	[46]
HDL/apo A–I	Circulation	NA	HDL inhibits PGRN convertion into GRN	[47]
SLPI	Circulation	NA	Convertion of PGRN into GRN	[17]
Tat (of HIV-1 and -2 and CAEV)	Circulation	GRN B + A	Unknown	[48, 49]
TNFR1 and -2	Membrane	GRN F + A + C	Inhibition of TNF-[α] signaling	[42]
TLR9	Membrane	GRN ACDE	Assisting the binding of CpG to TLR9	[44]
Sortilin	Membrane	GRN E	Delivery of PGRN to lysosome for degradation	[50]
Hexokinase	Cytoplasm	NA	Unknown	[51]
TPM3	Cytoplasm	NA	Promotion of hepatocarcinogenesis	[52]
BiP, calreticulin, GRP94, ERp57, ERp5, HSP70	ER	NA	Chaperones networking	[53]
Cyclin T1	Nuclear	GRN PFGBA DE	Inhibition of transcription	[54, 55]
Tat of HIV	Nuclear	GRN PFGBA DE	Inhibition of transcription	[54, 55]

NA, unknown.

As a novel ligand of TNFR, PGRN has become a significant, if not critical, molecule in the cubic of immunity, infection, and inflammation. In this review, we will present a summary of the functions of PGRN in the immune system, along with providing an illustration of its role in infection and inflammation. To avoid confusion, the abbreviated term PGRN is assigned to the full-length precursor of GRN, whereas GRN stands for the digested small peptides.

EXPRESSION AND FUNCTION OF PGRN

PGRN is highly expressed in epithelial cells, certain types of neurons, and macrophages [21] and is likewise expressed in a broad range of other tissues and cell types, including skeletal muscle, chondrocytes, adipose tissue, hematopoietic cells, and immune cells, including T cells and DCs [22–24]. PGRN has multiple physiological functions and is involved in many types of disease processes, including autoimmune disorders, cancer, and neurodegenerative diseases. PGRN, as a growth factor, promotes cell proliferation and is crucial to the development and generation of fast-growing epithelial, endothelial, and cancer cells [25]. PGRN levels are elevated in many types of cancer, including breast cancer, ovarian cancer, and cholangiocarcinoma [26–28]. The function of PGRN in cancer has been well-discussed and is not the topic of this review. Macrophage-derived PGRN is a key regulatory factor in the processes of inflammation and wound healing [29, 30]. Mutations of the PGRN gene are known to lead to the development of FTLD [31, 32], and the protein levels of PGRN in serum and cerebral spinal fluid were reduced significantly in unaffected and affected PGRN mutation carriers in FTLD [33–35]; thus, it can be postulated that measuring serum levels of PGRN may be applied in early diagnosis and detection of FTLD caused by PGRN null mutations. PGRN expression is increased in activated microglia in many neurodegenerative diseases, including Creutzfeldt-Jakob disease, motor neuron disease, multiple sclerosis, and Alzheimer's disease [31, 36–39].

PGRN inhibits myotube formation by reducing the activity of MyoD and up-regulating antimyogenic factor JunB [22], whereas it can also promote differentiation and proliferation of chondrocytes [40, 41]. PGRN null mice display skeletal defects, and administration of PGRN stimulates chondrocyte differentiation from mesenchymal stem cells in vitro and endochondral ossification ex vivo through the Erk1/2 signaling pathway [41]. PGRN also expressed in adipose tissues and is a key adipokine, mediating high-fat-induced insulin resistance, hence regulating the main pathological mechanisms of diabetes and obesity [23].

Aside from the noted functions across the organ systems, the importance of PGRN has also been emerging with respect to the immune system. Recently, PGRN has been reported to promote CD4⁺ T cells to differentiate into Foxp3⁺ Tregs [42]. PGRN has also been found to be highly expressed in a subpopulation of neutrophils that can promote antibody diversity in B cells [43]. PGRN also binds with TLR9 and assists in the recruitment of CpG-ODNs in macrophages [44], a process that is critical for the elimination of infection.

PGRN ACTIVITIES ARE TIGHTLY REGULATED BY THEIR ASSOCIATED PROTEINS

PGRN is a secreted glycoprotein, however it also functions at the cytoplasmic and nuclear level. So far, there are over 20 proteins that have been reported to bind with PGRN (Table 1). In the ECM, PGRN binds with ADAMTS7/12 [45, 56] and COMP [40]. PGRN inhibits ADAMTS7/12-mediated degradation of COMP. It binds with the EGF-like domain of COMP, and COMP, in turn, potentiates PGRN-stimulated chondrocyte proliferation [40]. The Perlecan domain V was associated with PGRN through its first two laminin- and EGF-like repeat domains [46]. Interaction between Perlecan domain V and PGRN contributes to a fine regulation of tumor angiogenesis and could ultimately affect cancer growth. PGRN also presents with binding activity between soluble proteins in the serum. For example, HDL/apo A–I have been noted to bind with PGRN and inhibit the conversion of PGRN to GRN in macrophages [47]. In another study, Tat proteins in HIV-1/2 and CAEV were also bound with PGRN, with their EGF-like repeat domains [48, 49]. Also, SLPI have been demonstrated to bind with PGRN in the cell culture medium and BAL, and SLPI inhibits the inflammatory response by protecting the cleavage of PGRN to GRN [17].

PGRN has also presented with features of binding with membrane receptors. For instance, PGRN binds with TNFR1 and TNFR2, as well as TLR9 [42, 44], both of which exhibit vitally important contributions to antigen presentation and T cell function, the aspects of which will be discussed later in this review. Sortilin has also been presented to bind with PGRN, forming an endocytosis-mediating complex, which plays a central role in the pathophysiology of frontotemporal degeneration [50]. However, another group shows that PGRN-mediated neuronal outgrowth is independent of Sortilin [57], suggesting that other receptors may mediate PGRN function in the nervous system.

PGRN has also been found to be expressed intracellularly. In the cytosol, PGRN binds with cytoplasmic proteins, including with hexokinase [51] and TPM3 [52]; however, the biological significance of this interaction is still unknown. PGRN also binds with ER chaperones, including BiP, calreticulin, GRP94, ERp57, ERp5, as well as HSP70, which is a substrate of protein disulfide isomerase family members [53]. In the cell nucleus, PGRN binds with cyclin T1 and Tat proteins of HIV to regulate gene transcription and inhibits HIV promoter-mediated transcription [54].

It is very unique that PGRN binds with a large variety of proteins at different levels, ranging from extracellular fluid and ECM to intracellular components, including the cytoplasm and nucleus. This versatility is probably a result of the special structure of PGRN, which consists of 7½ GRN domains and forms a string-bead-like structure. The combination of GRN domains may form distinctive structures that may be recognized by different proteins. For instance, the PGRN F–A–C domain forms a trimer-like configuration and binds with TNFR1/2 [42], and F–B domain is recognized by Laminin G and the EGF-like domain of Perlecan domain V [46]. Certainly, the multiple functions of PGRN are mediated by various associate proteins. However, it is unclear how PGRN conformation is acheived as to facilitate its binding with different partners in a tissue-specific or disease-specific manner.

Most of these associated proteins have been well-reviewed by Louis De Muynck and Philip Van Damme [58]. In this review, we will focus on how PGRN functions in the immune system through interplays with its associated proteins.

PGRN REGULATION OF THE IMMUNE SYSTEM

The function of PGRN in the immune system has not been clearly identified and elaborated on. HLA-DR is a MHC class II molecule and generally binds and presents peptides derived from endocytosed and processed exogenous antigens. It has been shown that in purified HLA-DR molecules from the B lymphoblastoid cell line, nine endogenous peptides were bound to HLA-DR11 αβ dimers, and a peptide from GRN D (AA41–56) was one of them [59]. This indicates that PGRN is involved in the antigen-presenting process; however, it is not clear how PGRN affects the structure and function of the MHC class II molecule.

A recent study has shown that PGRN is very important to antigen presenting in macrophages by binding with TLR9 [44]. TLRs are a class of proteins that play a key role in the innate immune system. They recognize structurally conserved molecules derived from microbes; for example, TLR3 recognizes polyinosinic:polycytidylic acid, TLR4 recognizes LPS, and TLR9 recognizes CpG-ODN DNA [60]. Depletion of PGRN impairs the response of TLR9 to CpG-ODNs, and as a result of binding with CpG-ODNs, also plays a role in intracellular delivery of CpG-ODNs to macrophages. It was depicted that CpG-ODNs strongly bound with WT macrophages but not with PGRN−/− macrophages, and an addition of purified PGRN in the culture medium restored the ability of PGRN−/− macrophages to acquire CpG. All of these results show that PGRN is critical in innate immunity against micro-organisms.

PGRN is highly expressed in CD8⁺CD28⁻ T cells rather than CD8⁺CD28⁺ T cells in humans [61]. CD8⁺CD28⁻ T cells, also called T-suppressor cells, maintain a regulatory function in many autoimmune diseases. For example, a study has shown that transferring CD8⁺CD28⁻ T cells suppressed experimental autoimmune encephalomyelitis [62]. Expression of high levels of PGRN in CD8⁺CD28⁻ T cells suggested that PGRN may be important in regulating the immune reaction. This hypothesis was also supported by another discovery. In the in vitro T cell differentiation experiment, PGRN induced naïve CD4⁺ T cells to specifically differentiate into CD4⁺Foxp3⁺ Tregs but not to Th1, Th2, and Th17 populations. PGRN also protected Tregs from negative regulation by TNF-α [42]. These findings suggest that PGRN may enhance the function of Tregs, which in turn, inhibits autoimmune diseases.

Our recent data show that PGRN−/− mice exhibit a decreased Th2 response in an OVA-induced asthma model and presented with decreased levels of IL-4, IL-5, and IL-13 and also decreased cell infiltration in BAL specimens (unpublished results). These findings indicate that PGRN promotes the Th2 response and is probably dependent on an interaction between DCs and T cells. PGRN was also shown to induce expression of Th2-like cytokines, IL-4 and IL-5, in neurons in another study [63].

PGRN REGULATION OF INFECTION

Neutrophils are critical for host defense and regularly and promptly recruit to the sites of bacterial invasion, generating large quantities of ROS [64] and releasing granular contents to kill pathogens [65]. PGRN is highly expressed in neutrophils, and it converts into GRNs by neutrophil-released elastase [17]. After cleavage, GRN B stimulates IL-8 expression in epithelial cells to recruit additional neutrophils to the site of inflammation. SLPI binds with PGRN and inhibits PGRN conversion to GRN by elastase, providing a switch to control innate immunity and inflammation [17].

The importance of PGRN in innate immunity is also observed in PGRN−/− mice infected with Listeria monocytogenes. Although macrophages from PGRN−/− mice express high levels of proinflammatory cytokines, few macrophages were found in the infected organs of PGRN−/− mice. Also, PGRN−/− mice failed to clear bacteria efficiently compared with WT mice [29]. These results demonstrate that PGRN is essential to processes countering the effects of infection through the regulation of innate immunity.

PGRN expression was induced significantly in gastric epithelial cells after infection with Helicobacter pylori [66, 67]. The induction required direct contact between epithelial cells with live H. pylori, while heat-killed bacteria were not able to induce PGRN expression. This suggests that increased PGRN expression is a natural response of gastric epithelial cells in response to infection [66]. However, it is still not clear whether increased expression of PGRN acts as a protective response to clear infection of H. pylori or whether it constitutes an adaptive reaction of the gastric epithelial cells. When treating Staphylococcus hyicus infection-related locomotor apparatus diseases in farm pigs, GRN injection in the knee joint, along with antibiotics, has been shown to be most effective [68].

PGRN also binds with Tat proteins of HIV-1 and HIV-2 and CAEV [48, 49], a lentivirus of goats that leads to chronic mononuclear infiltration of various tissues. Dimeric GRN A–B is a minimum requirement of binding with the cysteine-rich domain of the Tat proteins of HIV-1 and HIV-2 [48]. Besides regulating HIV transcription and replication, Tat protein is also secreted from infected cells and serves as an extracellular growth factor [69]. Cyclin T1 is a constituent of the transcription elongation factor P, which binds the HIV-1 transactivator Tat [54]. PGRN seems to play a protective role in HIV infection, as PGRN interacts with cylin T1, represses expression from the HIV promoter in transfected cells, and similarly represses transcription from cellular promoters [54, 55, 70]. Tat is also released to the extracellular environment from infected cells to repress the immune reaction by modulating major cytokines and membrane receptors. Tat stimulates expression of TGF-α and -β, the major cytokines inhibiting the immune reaction [71, 72]. Tat reduces expression of IL-2 and IL-2Rs in T cell lines, along with the expression of TNFR2 in the B cell line [73–75]. So far, it is unknown what the function of PGRN is when bound with extracellular Tat proteins.

PGRN REGULATION OF THE INFLAMMATORY RESPONSE

Having discussed the findings of the properties of PGRN in anti-infection, innate immunity, and immune regulation, it is necessary to present its intricate involvement in the inflammatory response with its recruitment into sites of inflammation and competition between inflammatory mediators. Although PGRN is one of the major anti-inflammatory molecules, the exact function of PGRN may vary depending on the stage and components involved in inflammation. For instance, PGRN acts by increasing the accumulation of neutrophils, macrophages, blood vessels, and fibroblasts in acute skin injury [30], but in acute infection, PGRN inhibits LPS-mediated IL-6, TNF-α, and MCP-1 cytokine release from macrophages [29]. The same is true for chronic inflammation. PGRN has exhibited a protective role in inflammatory arthritis [42] and IBD (see later, unpublished results), but PGRN is the major adipokine of mediated high-fat diet-induced insulin resistance by inducing up-regulation of IL-6 expression [23]. In this section, we will discuss the diverse functions of PGRN in acute and chronic inflammation in various disease entities, as well as their mechanisms, and the therapeutic application of PGRN and its derivatives.

The role of PGRN in acute inflammation

The acute inflammatory cascade involves the complex interplay and recruitment of humoral and cell-mediated, immunologic-generalized systemic responses to aid in the host's reaction to an acute and immediate presentation of foreign antigens, including those of viral, bacterial, or entirely exogenic origin, in the vital milieu [76]. The role of PGRN in the mediation of infectious acute inflammatory states has been elaborated on in the prior section, however the research conducted on the function of PGRN in tissue injury constitutes an integral component in the clarification of its key mechanisms and molecular protein interactions. It is widely known that with the acute onset of injury and discontinuation of tissue integrity, a cascade of biochemical signals propels forward a surge of factors initiating the characteristic “text-book” macroscopic clinical features of dolor (pain), calor (heat), rubor (redness), tumor (swelling), and functio laesa (loss of function). In acute skin injury, it has been shown that the expression of PGRN is induced immediately and remains at elevated levels up to 10 days [77]. Administration of PGRN to freshly made, transcutaneous puncture wounds increased the cell counts of fibroblasts, neutrophils, and macrophages but not lymphocytes [30]. PGRN also promotes tubule-like structure formation of human dermal microvascular endothelial cells [78]. It has also been demonstrated to enhance the acute wound-healing process in SLPI−/− mice, which have an inherently impaired healing mechanism as a result of elevated elastase levels, causing degradation of PGRN to GRN [17].

PGRN is also reported to be involved in acute neuronal injury [79]. PGRN as well as GRN and its intermediary fragments were significantly induced after injury of the spinal cord or dorsal root ganglia [80]. Induction of PGRN was noted mainly in the ipsilateral dorsal and ventral horn neurons and in activated microglia [80]. PGRN−/− mice showed intensified nociception and impaired motor function recovery [80, 81]. In the spinal cord contusion model, PGRN was induced in activated microglial cells in a time-dependent manner, reached peak concentration levels at 7 days following injury, and decreased gradually on subsequent days [82].

One mechanism by which PGRN promotes tissue repair is to increase cell proliferation and migration, executed through the PI3K and ERK MARK pathways. It has been identified that the pathways' specific inhibitors wortmannin and PD98059 significantly reduced PGRN-mediated cell migration in wounded skin tissues [30]. Both PI3K and MAPK pathways are very conservative in mediating the signaling of PGRN in many tissues, including fibroblasts endothelial cells, chondrocytes, and many types of cancer cells [30, 41, 83, 84]. Another mechanism involves the conversion of PGRN into GRN B, which stimulates epithelial cells to express IL-8, a chemokine that recruits neutrophils and macrophages to initiate tissue repair [17].

Contrastingly to physical injury, PGRN maintains a vital protective role in LPS-induced acute inflammation. LPS exposure was noted to trigger acute pulmonary inflammation with significant neutrophil infiltration in the BAL and increased expression of a panel of proinflammatory cytokines, such as TNF-α, IL-1β, IL-6, CXCL2, JE (the murine homolog of human CCL2), and KC (the murine homolog of human IL-8). Administration of PGRN has been shown to significantly prevent body weight loss and mortality, as well as reduce levels of proinflammatory cytokines in a dosage-dependent manner [85]. PGRN-mediated protection depends on TNFR2, as the TNFR2 antibody, but not the TNFR1 antibody, blocks the therapeutic effects of PGRN [85].

In another acute inflammation model, macrophages from PGRN-null mice, when treated with LPS, showed a significantly stronger induction of proinflammation cytokines, including IL-6, TNF-α, and MCP-1, than macrophages from WT mouse [29]. The anti-inflammatory role of PGRN seems to be mediated through IL-10, as PGRN-null macrophages fail to induce IL-10 expression after LPS treatment, and also, rPGRN and LPS costimulation synergistically induces IL-10 expression [29]. PGRN has neuronal protective effects in the acute response to ischemia. PGRN transgenic mice have a smaller degree of infarction as compared with WT mice in a middle cerebral artery occlusion model. Glial cells that overexpressed PGRN were protected from LPS-induced cytotoxicity in vitro. After LPS treatment, the expression of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) was much lower in glial cells from PGRN-overexpressing mice compared with glia from WT mice, whereas the expression of the anti-inflammatory cytokine IL-10 was up-regulated in glial cells from transgenic mice [86].

In acute injury and inflammation, PGRN seems to be important in the initiation of inflammation by recruiting fibroblasts and macrophages to the site of injury [87]. In the meantime, PGRN also functions in the mechanism of limiting the strength of inflammation by controlling the production of proinflammatory cytokines, such as IL-6, TNF-α, and MCP-1, through the up-regulation of IL-10.

The role of PGRN in chronic inflammation

Chronic inflammation, or the prolonged sustenance of the inflammatory state, is often caused by uncontrolled immune reactions, dysregulated, proinflammatory cytokine production, and the persistence of pathogens. The function of PGRN in chronic inflammation also depends on the pathophysiological context of the disease that it directly affects.

RA is a chronic autoimmune disease, which is mainly mediated by TNF-α [88–90] and is characterized by inflammation of synovial tissue, leading to progressive damage, erosion of adjacent cartilage and bone, and chronic disability. There are two membrane receptors of TNF-α: TNFR1 and TNFR2. TNFR1 is widely expressed in most cell lines and primary tissues, although TNFR2 is preferentially expressed on cells of the hematopoietic lineage [91]. TNF-α activates the caspase pathway and NF-κB pathway to induce apoptosis and expression of proinflammatory cytokines through TNFR1 and activates the AP-1 and NF-κB pathway to promote proliferation through TNFR2 (reviewed in ref. [92]).

Recently, PGRN was discovered as a novel ligand of TNFR1 and TNFR2 receptors. PGRN blocks TNF-α-mediated signaling pathways by competing with TNF-α binding to TNFR1/2 [42]. Administration of rPGRN significantly alleviated the symptoms of inflammatory arthritis in TNF-α transgenic mouse, as well as other RA animal models [42].

TNF-α was also the main mediator of IBD and other chronic autoimmune disorders, including ulcerative colitis and Crohn's disease, in human patients. Our recent results show that PGRN significantly expressed in the epithelium and muscular layers of colon tissue in WT mice in the course of dextran sulfate sodium or 2,4,6-trinitrobenzene sulfonic acid-induced colitis. In this study, a transfer of PGRN−/− CD4⁺CD45RB^hi T cells into RAG1−/− recipient mice promoted rapid development of disease and exacerbated transmural inflammation. Results indicated that CD4⁺CD45RB^hi T cell-derived PGRN was an essential component in reducing the signs of IBD. In the meantime, rPGRN displays a protective function in the mentioned colitis models (unpublished results).

Another example of the inverse relationship of PGRN and TNF-α levels involves studies concerning the development of bone demineralization, mainly, osteopenia [93]. It has been found that osteopenic phenotypes expressed significantly lower levels of PGRN, as well as of IL-13 and IL-10, and notably higher levels of TNF-α, IL-17, and IL-6. Increasing PGRN levels resulted in an augmentation of concentrations of IL-13, IL-10, and 25-(OH) vitamin D, as well as a decrease in TNF-α and IL-17 concentration [93]. Therefore, the action of PGRN also plays a significant role in osteocytic metabolism via mechanisms involving TNFR signaling.

Therefore, it can be speculated that the balance between PGRN and TNF-α is very critical to the initiation and progression of both chronic autoimmune disorders. As quantitative PGRN levels increase, the molar ratio of PGRN to TNF-α increases significantly during mutual competition for endogenous receptors TNFR1 and TNFR2; thus, the molar ratio of PGRN/TNF-α may be a good marker for the prognosis of autoimmune diseases. Advancement in potential therapeutic applications of the anti-inflammatory actions of PGRN included the creation of a PGRN derivative composed of one-half units of GRNs A, C, and F, plus linkers P3, P4, and P5, which retained binding affinity to TNFR [42]. This molecule was referred to as Atsttrin [94]. Both human rPGRN and Atsttrin produced a diminution of serum concentrations of proinflammatory cytokines IL-1β and IL-6 and COMP degradative fragments and increased concentrations of anti-inflammatory cytokines IL-10 and IL-13 when compared with the control [42]. These actions were tissue-specific and were studied in tibio-talar joint cartilage in RA animal models; however, interestingly, similar inhibitory actions were noted in IBD models.

Aside from antagonistic effects, precise regulation of PGRN also encompasses a mechanism that directly and parallelly increases PGRN and TNF-α levels. With predisposing molecular conditions, TNF-α expression may be up-regulated in the presence of PGRN [23]. The mutual codependency of these molecules has been studied in lipid metabolic pathways and inflammatory processes leading up to hypercholesterolemia and atherogenesis. It was presented that human embryonic kidney 293 cells transfected with PGRN increased the TNF-α and IL-1β expression in macrophagic cells; however, PGRN production was inhibited by cross-reacting with HDL or apo A–I, implicating its role as a lipoprotein [47]. The placement of antibodies directed against PGRN produced a decline of TNF-α and IL-1β on macrophages [47]. It was postulated that the conversion products of PGRN could be directly responsible for the augmentation in TNF-α and IL-1β levels, and HDL was believed to suppress the conversion of PGRN to GRN, hence inhibiting its proinflammatory effects.

In contrast to its anti-inflammatory role, PGRN also has proinflammatory effects in obesity and insulin-resistant diabetes mellitus [95]. PGRN serum concentrations were significantly higher in individuals with type 2 diabetes compared with normal or impaired glucose tolerance in obese subjects with predominant visceral fat accumulation. Serum PGRN significantly correlates with BMI, macrophage infiltration in omental adipose tissue, serum CRP concentrations, HbA1c values, and total cholesterol. Multivariable linear regression analyses revealed CRP levels as the strongest independent predictor of circulating PGRN [96]. The extent of in vitro PGRN-mediated chemotaxis is similar to that of MCP1 [96]. Another study has also shown that PGRN levels, as well as macrophages in omental adipose tissue, omental adipocyte size, and serum CRP, are significantly higher in insulin-resistant obesity than in insulin-sensitive obesity [97]. Moreover, PGRN gene expression demonstrated a significant correlation with BMI, visceral fat, MAPK, and AKT gene expression. The increased mass of visceral fat in correlation with the increased PGRN levels was more pronounced in the high or normal RMR group compared with the group with a low RMR. After adjusting for BMI and gender, it was found that circulating PGRN can predict the RMR/kg, independent of other variables, such as triglycerides, HDL, and CRP [95].

Consequently, PGRN was found to be a major adipokine to mediate high-fat-induced insulin resistance [23]. Expression of PGRN was induced by TNF-α in adipocytes, and the induction was blocked by a peroxisome proliferator-activated receptor γ agonist, which improves insulin resistance. A high-fat diet caused insulin resistance and significantly up-regulated expression of PGRN in fat tissues as well. PGRN−/− mice but not WT mice failed to develop insulin resistance when fed with a high-fat diet. Moreover, administration of PGRN caused insulin resistance in WT mice fed with a standard diet [23]. IL-6 was identified as a downstream cytokine to mediate PGRN-induced insulin resistance, as induction of IL-6 by a high-fat diet was totally blocked in PGRN−/− mice, and neutralizing IL-6 via antibody improves PGRN-induced insulin resistance [23].

Although circulating PGRN induces insulin resistance in adipose tissues, hypothalamic- derived PGRN is a glucose sensor to repress feeding. PGRN was highly expressed in periventricular tanycytes and in hypothalamic neurons, and its expression was decreased in a low-energy diet and increased in a high-energy diet. Intracerebroventricular administration of PGRN significantly suppressed nocturnal feeding. Moreover, the inhibition of hypothalamic PGRN expression increased food intake and promoted weight gain [98]. Although it is not clear how PGRN senses the level of glucose, it is speculated that it may enzymatically implement this through hexokinase, as previously, PGRN was found to form a complex with hexokinase with an unknown function [51].

Besides its involvement in glucose regulation, PGRN also demonstrates proinflammatory functions in NAFLD [99]. Serum PGRN levels are significantly higher in NAFLD than in the control group, and the serum PGRN levels were associated with lipid levels and the degree of hepatic fibrosis. After adjustment for potential confounders, serum PGRN remained to be an independent predictor of the degree of hepatic fibrosis in NAFLD patients [99].

PERSPECTIVES

With the use of a functional genomic approach and mass spectrum identification of purified protein complexes combined with biochemistry, cellular biology, and molecular biology techniques, the PGRN growth factor has been isolated and shown to physically associate with ECM proteins [40, 46], proteolytic enzymes [16, 17, 45, 56], and cell-surface receptors [42, 50]. So far, >20 PGRN-binding proteins have been reported. PGRN and its binding partners constitute interplay networks in mediating the pathogenesis of various pathophysiological processes, including immunity, infection, and inflammation. It is conceivable that novel PGRN-binding proteins will be identified in different tissues or under various physiological or pathological conditions.

Although the binding of PGRN to TNFRs was first identified using a yeast two-hybrid screen and subsequently confirmed through several in vitro and in vivo binding assays [42, 94, 100, 101], the exact molecular mechanisms and determinants underlying the interaction between TNFR and PGRN, as well as the PGRN-derived protein Atsttrin, still remain to be delineated. It appears that the correct conformation and/or oxidation states of PGRN rich in cysteine are critical for the binding of PGRN to TNFR, as evidenced by the fact that some commercially available rPGRNs exhibited less or no TNFR binding activity in an ELISA-based in vitro-binding assay (unpublished results), although they may retain other TNFR-independent activities.

It is well-established that PGRN activates ERK and PI3K/AKT pathways in several types of cells, such as chondrocytes [41]; however, its signaling pathways and the dependence on TNFR in immune cells remain largely unknown. PGRN binds TNFR2 with high-affinity, and Tregs constitutively express high levels of TNFR2 but do not express TNFR1 [102–104]. Thus, Tregs provide an excellent cell model to investigate PGRN/TNFR-mediated protective and beneficial signaling pathways in autoimmune diseases. In addition to TNFR2, PGRN binds with other receptors, including TLR9 [44, 105] and Sortilin [106] (Fig. 1). It is important to elucidate what signaling pathways and activities of PGRN are mediated by the individual receptors.

Figure 1. A diagram illustrates PGRN-mediated signal pathways through its binding receptors.

PGRN binds to TNFR1 and blocks TNF-α-induced inflammatory and apoptotic pathways. The binding of PGRN to TNFR2 may trigger an unknown protective signal cascade and promote Treg differentiation and function. PGRN assists to recruit CpG-ODNs to TLR9 in the endosome to enhance innate immunity and to protect the body from bacterial infection. Interaction between PGRN and Sortilin is important for PGRN trafficking and lysosomal degradation in neurons.

In the nervous system, the WNT signaling pathway is elevated in PGRN−/− mice [107], and targeting the WNT pathway has been proposed to be a potential strategy to treat PGRN mutation-induced frontotemporal dementia [108]. The WNT signaling pathway is also essential in the immune system, such as promoting T cell and B cell development, regulating the migration of peripheral immune cells, and playing a key role in modulating the function of APCs (reviewed in ref. [109]). It remains to be established and clarified whether the WNT pathway also counteracts with PGRN in the immune system and during inflammation.

Given that TNF signaling is known to be involved in >40 various kinds of diseases, and TNF inhibitors have been used to treat several kinds of autoimmune diseases, including RA and colitis, we envisage potential applications of PGRN, especially its derivative Atsttrin, in these and other autoimmune diseases, such as systemic lupus erythematosus, ankylosing spondylitis, plaque psoriasis, and psoriatic arthritis. PGRN and Atsttrin might also be useful in neuronal degenerative diseases, in which TNF is believed to play an important role in disease pathogenesis, notably FTLD, which is considered a chronic inflammatory disease.

Current TNF inhibitors, such as Remicade and Humria, target the TNF ligand, whereas PGRN and Atsttrin selectively target TNFR, and it is worthwhile to determine whether blockage of both ligand and receptors simultaneously will be more effective through comparing the effects of Atsttrin alone or with current TNF inhibitors. In addition, increasing animal and clinical data indicate that MTX or glucocorticoid improves the therapeutic effects of current TNF inhibitors; for instance, MTX was shown to enhance the response of patients to Humria treatment and to reduce Humria immunogenicity of patients [110]; thus, it is important to determine whether MTX will increase the effectiveness of Atsttrin in treating various kinds of autoimmune diseases.

DISCLOSURES

C.L. is the cofounder and shareholder in Atreaon.