The multifaceted roles of neutrophil gelatinase associated lipocalin (NGAL) in inflammation and cancer

Abstract

Neutrophil gelatinase associated lipocalin (NGAL), also known as oncogene 24p3, uterocalin, siderocalin or lipocalin 2, is a 24kDa secreted glycoprotein originally purified from a culture of mouse kidney cells infected with simian virus 40 (SV-40). Subsequent investigations have revealed that it is a member of the lipocalin family of proteins that transport small, hydrophobic ligands. Since then, NGAL expression has been reported in several normal tissues where it serves to provide protection against bacterial infection and modulate oxidative stress. Its expression is also dysregulated in several benign and malignant diseases. Its small size, secreted nature and relative stability have led to it being investigated as a diagnostic and prognostic biomarker in numerous diseases including inflammation and cancer. Functional studies, conducted primarily on lipocalin 2 (Lcn2), the mouse homologue of human NGAL have revealed that Lcn2 has a strong affinity for iron complexed to both bacterial siderophores (iron-binding proteins) and certain human proteins like norepinephrine. By sequestering iron-laden siderophores, Lcn2 deprives bacteria of a vital nutrient and thus inhibits their growth (bacteriostatic effect). In malignant cells, its proposed functions range from inhibiting apoptosis (in thyroid cancer cells), invasion and angiogenesis (in pancreatic cancer) to increasing proliferation and metastasis (in breast and colon cancer). Ectopic expression of Lcn2 also promotes BCR-ABL induced chronic myelogenous leukemia in murine models. By transporting iron into and out of the cell, NGAL also regulates iron responsive genes. Further, it stabilizes the proteolytic enzyme matrix metalloprotease-9 (MMP-9) by forming a complex with it, and thereby prevents its autodegradation. The factors regulating NGAL expression are numerous and range from pro-inflammatory cytokines like interleukins, tumor necrosis factor-α and interferons to vitamins like retinoic acid. The purpose of this review article is to examine the expression, structure, regulation and biological role of NGAL and critically assess its potential as a novel diagnostic and prognostic marker in both benign and malignant human diseases.

Figure 1. Schematic representation of the lipocalin fold

The characteristic feature of lipocalins is the “lipocalin fold” which comprises of an N-terminal 3–10 helix followed by eight beta sheets (A–I) arranged in an anti-parallel orientation. The eighth beta sheet is connected to an alpha helix (denoted as α1), which is in turn connected to a C-terminal beta sheet. The beta sheets are connected by loops (L1-L7). Loops L1, L3, L5 and L7 form the open end of the molecule (i.e. the opening to the ligand binding site of NGAL). The portion of the lipocalin fold that are structurally conserved between different lipocalins is indicated by the blue boxed regions while the region that shows significant conservation in amino acid sequence is indicated by the black boxed region.

Figure 2. Transcripts encoded by the human and mouse NGAL genes

The boxes represent exons while the connecting lines represent introns. Filled in boxes represent conding sequences, while empty (unfilled) boxes represent the untranslated region (UTR). The number above the transcript is the length of the mature transcript (indicated as number of base pairs). The number of amino acids corresponds to the number of residues that are translated. The length of each transcript is proportional to the length of the genomic DNA (Source: http://www.ebi.ac.uk)

Figure 3. Regulation of Lcn2 expression in macrophages

Macrophages break down senescent red blood cells (RBCs) releasing free iron from the hemoglobin contained within them. This free iron is then exported out of the macrophages by two pathways: a) by a transmembrane iron exporting protein ferroportin and b) as a complex with lipocalin 2 (Lcn2). Macrophages are also the site of infection by Salmonella, a gram negative intracellular bacteria. The bacteria require iron for their growth and survival. They express enterochelin, a siderophore on their outer membrane which binds to the free iron (released from the RBCs) and transports it into the bacterial cell. Infection of macrophages by Salmonella upregulates the expression of the anti-microbial protein lipocalin 2 (Lcn2). This binds to the iron-laden siderophores, thereby preventing the bacteria from using the free iron for their proliferation. This, together with the increased export of iron from the macrophages deprives bacteria of iron and induces growth arrest. The gene product of HFE (HFE) is a transmembrane protein similar to major histocompatibility antigen-1. It is expressed on the macrophage cell membrane and regulates both the uptake and efflux of iron from these cells. It is also a negative regulator of Lcn2 expression. Loss of functional HFE1 (most commonly by mis-sense mutations in patients with hereditary hemochromatosis) leads to an upregulation of Lcn2 in the macrophages thus conferring resistance to the host against infection by Salmonella.

Figure 4. Mechanism of bacteriostatic action of NGAL

Gram negative bacteria (like Salmonella typhimurium) trigger an immune response characterized by the activation of antigen presenting cells (macrophages and dendritic cells) upon engulfing the bacteria. These activated cells then release cytokines like IL-18 and IL-23 that in turn activate T-lymphocytes. These activated T-cell in turn release IL-17 and IL-22. These two cytokines act on the intestinal cells and stimulate the de novo synthesis of lipocalin 2 (Lcn2). The Lcn2 is secreted into the intestinal lumen and binds to bacterial iron binding proteins (siderophores) like enterochalin. Since iron is essential for the growth of bacteria, the sequesteration of bacterial siderophores by Lcn2 has a bacteriostatic effect. In some gram negative bacteria (like Salmonella typhimurium), a cluster termed as the iroBCDEiroN cluster is present which encodes for salmochelin, a gycosylated derivative of enterochelin which does not bind Lcn2. Hence, gram negative bacteria containing this cluster are resistant to the bacteriostatic effects of Lcn2. Commensals like certain species of E.coli lack the iroBCDEiroN cluster and hence are targeted by Lcn2 secreted during intestinal inflammation.

Figure 5. Regulation of NGAL expression by NF-κB

NF-κB is a dimer composed of one or more of five members of the NF-κB family (p50, p52, p65, Rel-A, Rel-B or c-Rel). Under resting state, the complex remains inactive in the cytoplasm bound to the inhibitor of IκB (IκB α or β). (A) NGAL inducers like IL-1β activate the I-kappa kinase (IKK) which phosphorylates IκB. This promotes ubiquitylation and subsequent degradation of IκB, thus freeing the NF-κB complex. (B) This complex now translocates to the nucleus where it upregulates the expression of the co-activator IκB-ζ (through a κB response element on its promoter) (Broken yellow arrows). This cofactor then binds to the NF-κB dimer (in the nucleus) and the complex in turn binds to the κB response element (κB RE) located at position −181/−171 on the NGAL promoter. This leads to activation of transcription of NGAL. IκB-ζ itself however, does not bind to the κB-RE on the NGAL promoter. (C) Incidentally, the NF-κB inhibitor IκB also has a κB RE on its promoter. Thus, its expression is also paradoxically upregulated by NF-κB. IκB in turn binds to the NF-κB transcription complex and transports it out of the nucleus (D), thus terminating its transcriptional activity. (E)TNF-α on the other hand binds to TNF-receptor (type 1 present in most tissues or type 2 present only on immune cells), which activates the TNF receptor type 1 associated DEATH domain protein (TRADD in case of TNFR-1). TRADD then recruits TRAF2 (TNF receptor associated factor) and the serine threonine kinase RIP (receptor interacting protein). TRAF2 also recruits IKK (existing as a complex of IKKα,β and γ) which is phosphorylated (and thus activated) by RIP. IKK then phosphorylates and thus targets IκBα for degradation. However, unlike IL-1β, the activated NF-κB induced by TNF-α does not induce transcription of the co-factor IκB-ζ and thus does not induce NGAL expression upon its transport into the nucleus. The differential effect of TNF-α and IL-1β on IκB-ζ is suggested to occur by increased stabilization (and therefore translation) of the IκB-ζ mRNA by IL-1β (but not TNFα). (F) TLR signaling and NGAL regulation. Toll-like receptors (TLRs) are a family of evolutionary conserved proteins that function as part of the body’s innate immune system defense mechanism to recognize pathogen associated molecular signatures. Ten TLR proteins have been identified so far in humans, each of which has a specific ligand. Activation of TLR signaling occurs in response to the binding of specific ligands (microbial lipopeptides for TLR2 and lipopolysaccharide of gram negative bacteria for TLR4). Myeloid differentiation primary response gene (MyD88), an adaptor protein used universally by all TLRs for signaling, possesses a C-terminal Toll/IL-1 receptor (TIR) domain that binds to the cytoplasmic portion of the TLRs. Following this, the IL-1 receptor-associated protein kinase (IRAK) proteins (IRAK-1 and 4) are recruited to the TLRs by interaction between the death domains of MyD88 and IRAK. IRAK-1 is then phosphorylated (activated) and in turn associates with the TNF receptor associated factor 6 (TRAF6). The IRAK-1/TRAF6 complex then dissociates from the TLR receptor and associates with TGF-beta activated kinase 1 (TAK1) and the TAK1 binding proteins (TAB1 and TAB2). Till this step the complex is attached to the cell membrane. The IRAK-1 protein is then degraded allowing the TRAF6/TAK1/TAB1/TAB2 complex to move from the cell membrane into the cytoplasm where it associates with, among other proteins with the E3 ubiquitin ligases (Ubc13 and Uec1A). The ubiquitin ligases catalyze the synthesis of a Lys-63 linked polyubiquitin chain on TRAF6. This induces TRAF6 to activate TAK1 which in turn phosphorylates the IKK complex (IKK a,b,g) or the MAP kinases (e.g. JNK) thus leading to transcription of NF-kB and AP-1 regulated genes respectively (including NGAL).

Figure 6. Dual regulation of NGAL expression by EGFR

(A) In pancreatic cancer cells, treatment with recombinant EGF results in a downregulation of both NGAL mRNA and protein. The mechanism involves upregulation of Zeb1, a transcription factor that inhibits E-cadherin synthesis. E-cadherin helps maintain NGAL expression in pancreatic cancer cells by activating NF-kappaB mediated transcription of NGAL. (B) In renal tubular epithelial cells, EGF treatment leads to a significant upregulation of NGAL mRNA and protein. This occurs through an upregulation of the transcription factor HIF-1 alpha, which in turn promotes transcription of NGAL. This induction of HIF-1 alpha is specific to EGF and independent of hypoxia.