INTRODUCTION
Leukocyte cell‐derived chemotaxin‐2 (LECT2) is a highly conserved protein with roles
in cell development, sepsis, liver disease, metabolic syndrome, arthritis, cancer,
and amyloidosis, among others. We will review its genetics, structure, role in physiology,
and its future as a biomarker and therapeutic target across many diseases. Studies
involving LECT2 were identified using PubMed and compiled into a single review paper.
Research on LECT2 suggests a future as a biomarker of disease and potential therapeutic
target.
BACKGROUND
The discovery of leukocyte cell‐derived chemotaxin‐2 (LECT2) was the result of a drive
to find novel biomarkers of inflammatory diseases.1 It was first isolated in humans
by Yamagoe et al.1 from phytohemagglutinin‐stimulated T‐cell leukemia SKW‐3 cells
and identified as a chemotaxin of neutrophils. Chemotaxins are substances released
by cells that stimulate the movement of white blood cells and LECT2 derived its name
from this in vitro chemotactic ability at its initial discovery.1 However, it is now
known to have several other functions in the human body and it is no longer limited
to chemotaxis. There has been a rapid expansion of knowledge regarding its function
with major developments in liver regeneration,2, 3, 4, 5, 6, 7, 8 immune modulation,1,
9, 10, 11, 12, 13, 14, 15 bone growth,9, 16, 17, 18, 19, 20 neuronal development,21
glucose metabolism and metabolic syndrome,10, 22, 23, 24, 25, 26, 27 cancer,9, 28,
29, 30, 31, 32, 33 and amyloidosis,34, 35, 36, 37, 38, 39 among others. It is often
found secreted in the bloodstream and expression of this protein has been identified
in liver cells, neurons, various epithelial cells, parathyroid cells, and white blood
cells, although it is felt to be most often secreted by the liver.40 However, despite
the multifunctional role of LECT2 in several different organ systems, there has yet
to be a comprehensive review that summarizes all of the current knowledge and information.
We will attempt to synthesize current reported literature on this increasingly important
protein and explore its potential future in modern clinical medicine in relation to
disease pathophysiology (Table
1)6, 13, 17, 18, 22, 28, 29, 33, 41, 42 and as a biomarker (Table
2).2, 3, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 22, 23, 24, 28, 29, 30, 31,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42
Table 1
Association of leukocyte cell‐derived chemotaxin‐2 with pathophysiology and disease
in different organ systems
Organ system
Findings
Ref.
Study description (subjects, sample size, study design)
Inflammation and fibrosis
LECT2 expression changes with signs of inflammation or fibrosis
40
In vivo human study using immunohistochemistry to study 28 different organs in the
human body (n = 68) for LECT2 expression in normal and diseased states
Immune system
Activated immune response and prosurvival role with infection and sepsis
15
In vivo and in vitro nonhuman study of healthy fish (n = 4) demonstrating increased
cytokine expression, induced chemotaxis, and monocyte / macrophage activation by LECT2
mediated by C‐type lectin receptor (PaCLR) that is inhibited by anti‐PaCLR antibodies
Activated immune response and prosurvival role with infection and sepsis
10
In vivo nonhuman study in mice using models of bacterial infection and cecal ligation
to show that LECT2 had a protective effect in mice through CD209a receptor interaction
to activate macrophages
Activated immune response and prosurvival role with infection and sepsis
12
In vitro nonhuman activity of fish macrophages and their response to recombinant LECT2
using qPCR, chemotactic activity assays, and polyacrylamide gel electrophoresis
Activated immune response and prosurvival role with infection and sepsis
13
In vivo human retrospective study of healthy controls (n = 31) and patients admitted
to the intensive care unit with confirmed sepsis (n = 23) comparing serum LECT2 to
outcomes, vital signs, routine bloodwork, and cytokines
Neutrophil chemotaxis
9
In vitro human study of neutrophil chemotaxis compared with fMLP
Bone
Involved in the pathogenesis of osteoarthritis and rheumatoid arthritis
16
In vivo nonhuman study in WT and LECT2‐deficient mice after induction of arthritis
by anti‐type II collagen antibodies and lipopolysaccharide (n = 7 in each group) as
assessed by hind paw thickness, histology, and cytokine expression. Confirmed by hydrodynamic
gene transfer of LECT2 into deficient mice to monitor improvement (n = 10 in each
group)
Polymorphisms of LECT2 associated with more severe forms of RA
17
In vivo human cross‐sectional study using DNA sequencing of adults with RA (n = 204)
and controls (n = 197) found that the LECT2 Val58Ile polymorphism was associated with
increased severity of disease in RA
Polymorphisms of LECT2 associated with more severe forms of RA
19
In vivo human cross‐sectional study using DNA sequencing of adults with RA (n = 105)
and controls (n = 101) found that the LECT2 Val58Ile polymorphism was associated with
increased severity of disease in RA
Expression of LECT2 associated with severity of disease in OA
18
In vivo human cross‐sectional study using isobaric tags for relative and absolute
quantitation and Western blot in patients with OA (n = 17) and controls (n = 6) to
identify LECT2 as a biomarker for severity of disease
Intestines
Loss of LECT2 function in mice can lead to deregulated Wnt‐signaling in mice and possibly
contribute to adenoma formation
31
In vivo nonhuman study of mice and small intestine adenoma formation involving the
APC and Wnt signaling pathways. WT (induced AhCre+ Apc+/+ Mbd2+/+) mice were compared
with Mbd2‐deficient (induced AhCre+ Apc+/+ Mbd2−/−) and Apcfl/fl (induced AhCre+ Apcfl/fl
Mbd2+/+)) and Apcfl/fl Mbd2−/− (induced AhCre+ Apcfl/fl Mbd2−/−) mice to assess for
changes in gene expression, adenoma formation, and changes in Wnt‐pathway signaling
Liver
Involvement in hepatic inflammation, injury, and recovery
2
In vivo nonhuman study of mice (n = 6) to assess for changes in LECT2 expression,
serum transaminase levels, and cytokine release following Con A induced hepatic injury
Assessment of the effects of LECT2 on SEA‐induced hepatitis
14
In vivo nonhuman study that compared B6 mice (n = 10) to LECT2‐deficient mice (n =
10) when exposed to SEA. Also assessed B6 mice response when treated with SEA as well
as LECT2 (n = 15) and not treated with LECT2 (n = 15)
Changes after liver transplantation
3
In vivo human study of living related donor liver transplant donors (n = 5) and recipients
(n = 5) where serum LECT2 and serum transaminases were trended over several days following
the procedure
Associated with acute liver failure survival
6
In vivo human study (n = 6) following adult patients with acute liver failure and
compared serum LECT2 levels with serum transaminases and outcomes
Upregulated and downregulated in certain human liver cancers
9
In vitro human study from samples of hepatoblastoma (n = 14) and hepatocellular carcinoma
(n = 15) using qPCR to assess for up and down regulation
LECT2 expression increased in mice with liver tumors
9
In vivo study of mice given an oncogenic form of β‐catenin with liver tumors (n =
15) showing increased LECT2 expression via Western blot, Northern blot, and immunohistochemistry
Deficiency of LECT2 increases hepatic NKT‐cells in mice worsening hepatitis
11
In vivo mouse study (n = 6 at each time point) comparing WT and LECT2 deficient mice
after Con A induced hepatitis to assess for cytokine production, histology of the
liver, and presence of immune cells
Tumor suppressor in certain HCCs
28
Multipart study using in vitro HCC cell lines and well as in vivo human and nonhuman
subjects. Human patients with HCC (n = 73) were assessed by immunoblotting found that
increased LECT2 suppresses MET phosphorylation and is associated with decreased vascular
invasion and improved survival. Mice were studied via orthotopic liver injection experiments
to assess the effects of loss of function LECT2 mutations on HCC. Cell lines were
used to identify the HxGxD as the binding motif for LECT2
Expression pattern of LECT2 changes with staging of HCC
42
In vivo human subject study of liver biopsies using immunohistochemistry to compare
the expression patterns of LECT2 in low‐grade malignant HCC (n = 9), advanced HCC
(n = 5), and atypical hyperplasia (n = 19)
Potential biomarkers in HCC
29
Multipart study using in vitro human cell lines and well as in vivo human and nonhuman
subjects. Human patients with HCC (n = 54) were compared with healthy controls (n
= 11) and patients with cirrhosis but no HCC (n = 16) by ELISA for LECT2, PCR to sequence
β‐catenin, and qPCR to assess expression. Microarray was used to assess for LECT2
regulation in WT and β‐catenin knockout mice. Human cell lines were then assessed
for β‐catenin regulation of LECT2 using qRT‐PCR, β‐catenin knockdown, and ChIP analysis.
A mouse model of HCC (n = 9) was assessed for β‐catenin mutations via direct sequencing
and then correlated with LECT2 expression via qRT‐PCR.
LECT2 is able to effect β‐catenin induced inflammation
30
In vivo nonhuman study of mice using inactivated APC type mice (to model tumor initiation)
and Lpk‐myc+ mice (to model tumor progression) to assess the inflammatory setting
of tumor development. These models were then cross bred with LECT2‐deficient mice
to assess the effects of LECT2 on the development of hepatic inflammation and tumor
development.
Abnormal folding associated with amyloidosis
35
In vivo human study of subjects with hepatic amyloidosis (n = 130) using histology
and microdissection / mass spectrometry to identify the prevalence of abnormally folding
LECT2
Abnormal folding associated with amyloidosis
38
In vivo human study of retrospective samples from adult patients with hepatic amyloidosis
(n = 70) to determine prevalence of abnormal LECT2 using immunohistochemistry and
microdissection / mass spectrometry as well as a second phase to assess histologic
morphology of abnormal LECT2 amyloidosis (n = 24)
Endocrine and metabolic systems
Increased levels of LECT2 are associated with increased insulin resistance
22
Study of both in vivo human and nonhuman subjects. Human subjects involved DNA chip
analysis of liver biopsies (n = 10 with type 2 diabetes and n = 7 healthy controls)
to correlate LECT2 mRNA and obesity. Then also used LECT2 enzyme linked immunosorbent
assays to correlate serum LECT2 with various markers of metabolic syndrome (n = 200).
Afterward, mice were used to demonstrate LECT2 increasing insulin resistance in skeletal
muscle via phosphorylation of Jun NH2‐terminal kinase and that LECT2 deletion increased
insulin sensitization and other markers of metabolic syndrome.
LECT2 as a therapeutic target in metabolic syndrome
23
In vitro study of human HCC cell lines and in vivo study of mice of the effects of
LECT2 and a dipeptidyl peptidase‐IV inhibitor (gemigliptin). HCC cell lines were assessed
for molecular markers of fatty liver disease after LECT2 administration as well changes
after gemigliptin administration via Western blot and histology. Mice fed a HFD without
treatment (n = 7), with treatment (n = 7), and lean controls (n = 7) were assessed
for body weight, glucose, and insulin tolerance tests, and Western blot for molecular
markers of insulin signaling.
LECT2 may have utility as a serum marker for obesity and NAFLD
41
In vivo cross‐sectional human study of Japanese adults (n = 231) using ELISA to measure
LECT2 and compare levels to anthropometric and clinical variables to assess utility
as serum biomarkers
Oncology
Prognostic indicator in breast cancer
33
In vivo human study of breast cancer tissue samples using microarray (n = 247) and
real time PCR (n = 98) to assess for gene expression and clinical data to develop
gene expression profiles that could predict clinical outcome
Renal
Abnormal folding associated with amyloidosis
34
In vivo human study of a single patient with renal amyloidosis using biochemical analysis
and immunohistochemistry to identify the abnormal folding of LECT2 as a novel cause
of renal amyloidosis
Abnormal folding associated with amyloidosis
36
Case series using human subjects with renal amyloidosis to compare demographic, clinical,
histologic features, and electron microscopy (n = 40), as well as genetic sequencing
of the LECT2 gene (n = 10)
Multiorgan system disease
Abnormal folding associated with amyloidosis
37
Human autopsy study with two phases to identify prevalence of abnormal LECT2 in amyloidosis
by immunohistochemistry. Initial phase was a review of autopsies between 2010 and
2012 of those who died over the age of 45 (n = 524) with a second more focused phase
of Hispanic (n = 376) and Native American Indians (n = 101).
Pulmonary
Abnormal folding associated with amyloidosis
39
In vivo human case report using immunohistochemistry to demonstrate abnormal LECT2
amyloidosis presenting as pulmonary‐renal syndrome with lung deposits in addition
to already described renal deposits
Vascular
Associated with atherosclerotic signaling
24
In vitro study of human umbilical vein endothelial cells and THP‐1 cells treated with
varying doses of LECT2 to assess for function and signaling pathways through Western
blot and qPCR
ChIP, chromatin immunoprecipitation; Con A, concanavalin A; ELISA, enzyme‐linked immunosorbent
assay; fMLP, N‐formylmethionine‐leucyl‐phenylalanine; HCC, hepatocellular carcinoma;
HFD, high‐fat diet; LECT2, leukocyte cell derived chemotaxin 2; Mbd2, methyl binding
domain protein 2; NAFLD, nonalcoholic fatty liver disease; NKT, natural killer T cells;
OA, osteoarthritis; PaCLR, Plecoglossus altivelis C‐type lectin receptor; PCR, polymerase
chain reaction; qPCR, real time polymerase chain reaction; qRT‐PCR, quantitative reverse
transcriptase‐polymerase chain reaction; RA, rheumatoid arthritis; SEA, staphylococcal
enterotoxin A; WT, wild type.
John Wiley & Sons, Ltd.
Table 2
Leukocyte cell‐derived chemotaxin‐2 as a biomarker of disease
Disease
Findings
Ref.
Acute liver failure
Potential prognostic indicator
6
HCC
Potential prognostic indicator
28, 29, 42
RA
Potential marker for severity of disease
17, 22
OA
Potential marker of grade and severity of disease
18
Metabolic syndrome
Potential marker of severity of insulin resistance and obesity
41
Sepsis
Potential prognostic indicator
13
Breast cancer
Potential predictor of recurrence and mortality in female smokers
33
HCC, hepatocellular carcinoma; OA, osteoarthritis; RA, rheumatoid arthritis.
John Wiley & Sons, Ltd.
METHODS
Research papers were identified using PubMed and Ovid Medline using the term “leukocyte
cell‐derived chemotaxin 2.” Papers identified were reviewed by all authors for relevance
to the subject matter and if they were published in peer‐reviewed journals. These
studies were also used to identify further publications not found in the PubMed search.
There were a total of 52 papers included in the study. Inclusion criteria included
any publication that directly studied LECT2 in vitro either in human or nonhuman studies,
and studies were excluded if they did not discuss LECT2 in the setting of either physiology
or disease. Included studies were found in various searches from May to October 2016.
References include publication years ranging from 1996 to 2016.
Protein
The protein was first purified and identified in humans from phytohemagglutinin‐stimulated
SKW‐3 leukemic T‐cells as a 16 kDa secreted protein with 133 amino acids in humans.1,
43, 44 At the time of discovery, the human protein structure was found to be 48% similar
to myb‐induced myeloid protein‐1 in chickens, 80% similar to mouse LECT2, and 86%
similar to bovine LECT2 (also known as chondromodulin‐II).1, 45 It has since been
isolated in various species from mammals to shellfish.12, 15 This suggests an important
regulatory function because it is relatively conserved across a wide variety of species.
It also suggests that a common ancestor of LECT2 was passed down several generations
and modified. The typical mouse LECT2 protein is 151 amino acids, although there is
also an atypical type that is smaller at 132 amino acids due to the early termination
signal from exon 4.46 The initial studies by Yamagoe et al.1 revealed in vitro chemotactic
properties of LECT2, which contributed to its name. Further studies have shown that
LECT2 can form various oligomers in physiologic conditions, but the noncovalent binding
of a zinc atom stabilizes the structure and prevents oligomer formation.47 There are
three disulfide bonds between six evolutionarily conserved cysteine residues.47 The
crystal structure of LECT2 was recently identified by Zheng et al.48 (Figure
1).
Figure 1
The crystal structure of human LECT2 identified by Zheng et al.
Although discovered in leukemic T‐cells, it was later identified in fetal livers,
adult hepatic cell lines, and as a secreted protein in the blood.49 Subsequent studies
have estimated its concentration in the bloodstream at ∼20 ng/mL in adult humans,
but this can vary depending on disease state.13 Prior to secretion, it is generally
found in the cytoplasm of cells. Using immunofluorescence, Yamagoe et al.1 demonstrated
that the LECT2 protein is found in the cytoplasm of human hepatocytes and is also
localized in some endothelial cells of hepatic arteries, portal veins, and central
veins prior to secretion.49
In terms of function, the structure of LECT2 protein is similar to the M23 family
of metalloendopeptidases, but it is unclear if they share any common functions. It
has also been found to be similar to bovine chondromodulin‐II, which is involved in
bone health. This has led researchers to investigate where else in the body LECT2
could be found.40, 43 Nagai et al.40 studied the LECT2 protein in various tissues
of the body. They found that LECT2 is generally expressed in vascular, endothelial,
and smooth muscle cells, adipocytes, cerebral nerve cells, apical squamous epithelia,
parathyroid cells, sweat and sebaceous glandular epithelia, Hassall bodies, and some
mononuclear cells in immunohematopoietic tissue. Additionally, in cells and tissues
where LECT2 is normally found, decreased expression is observed during tissue inflammation,
fibrosis, and pathology. This suggests that when LECT2 is a part of normal function,
the degeneration of tissue homeostasis can downregulate its expression.40 Furthermore,
tissues that generally did not have LECT2 expressed at baseline included osteoblasts,
chondrocytes, cardiac tissue, smooth muscle cells in the gastrointestinal tract, and
the epithelial cells of some tissues. In these tissues, LECT2 expression increased
during disease conditions. It appears that in cells where LECT2 is not a normal part
of cellular function, it is upregulated in disease settings and is likely to play
a critical role in pathobiology.40
Interaction of LECT2 with other proteins and receptors is still under investigation,
but some molecular pathways have been elucidated. For example, in hepatocellular carcinoma
(HCC), LECT2 has the ability to bind and inhibit hepatocyte growth factor/MET receptor
through protein tyrosine phosphatase 1B recruitment.28 It can also bind to CD209a
receptors in mice macrophages to affect the immune system.22 LECT2 has also been noted
to increase phosphorylation of Jun NH2‐terminal kinase (JNK) resulting in both insulin
resistance and atherosclerotic disease.10, 24
However, what we know about LECT2 pathways is eclipsed by what we do not know. The
M23 proteins are a group of the protease family and it would be easy to assume that
LECT2 has a similar role in the body. Despite this, there has been no substrate discovered
at this time for LECT2 protease activity.47 Either its substrate is very specific
and has yet to be discovered or there is no substrate and the function of LECT2 is
unrelated to its protease cousins. Using in vitro animal studies, LECT2 was found
to target osteoblasts and promote bone growth, but its target that causes this is
not known.50 The same applies to its in vitro chemotactic ability.1, 9 It is clear
that LECT2 interacts with a wide range of proteins and cells in the human body, but
before we can reliably target LECT2 as a potential biomarker or drug target, more
work will need to be done mapping out its pathways.
Genetics
Yamagoe et al.,1 the group that initially isolated the protein, mapped the LECT2 gene
to 5q31.1‐q32 in humans.43 This location is near several other immune‐modulating proteins,
including interleukin (IL)‐3/4/5/9 and granulocyte macrophage‐colony stimulating factor.43
The LECT2 gene is ∼8 kb and contains four exons and three introns.43 The gene is translated
as 1–1.3 kb mRNA with four different major transcription start sites and several minor
start sites.43, 51 Polymorphisms were quickly identified. The first identified was
at codon 58 in exon 3 that can code for either valine or isoleucine.43, 51 Subsequent
studies have found that this polymorphism, Val58Ile, is associated with worsening
joint destruction in rheumatoid arthritis (RA).17, 19 Additionally, there is a single
nucleotide polymorphism at position 172 that is implicated in renal amyloidosis from
LECT2.36 Other polymorphism and disease associations are still under investigation.
General population data for frequency of polymorphisms have not been published, to
the best of our knowledge. Definitive expression is still under investigation and
although mRNA is largely identified in the liver, further areas of expression continue
to be identified.49
Mouse LECT2 was first described by Yamagoe et al.46 and it is found on chromosome
13 and contains five potential exons with five major transcription start sites. However,
the commonly expressed mRNA does not contain exon 4 and is closer to the human product.
The gene occasionally includes exon 4 producing an atypical LECT2 protein through
a frameshift and results in early termination.46 It is thought that this atypical
mouse LECT2 has its own unique function in mouse physiology and does not have a human
counterpart.46
Gene regulation
Given that LECT2 has multiple functions across many organ systems, there is no one
clear molecular pathway that regulates LECT2 expression. LECT2 is associated with
liver regeneration and there is evidence that the β‐catenin / Wnt‐pathway directly
activates LECT2 expression.9 Wnt activation disrupts the normal function of cellular
destruction pathways leading to an accumulation of β‐catenin in the cytoplasm.52 The
β‐catenin then binds to the TCF/LEF family of transcription factors and binds to the
identified promoter regions of both mice and human Lect2 genes resulting in increased
expression.9 Additionally, methyl binding domain protein 2 (Mbd2) can also downregulate
expression by binding to its promoter region and is also part of the Wnt‐pathway.31
Interestingly, LECT2 has negative feedback on the same pathway that induces its expression
by inhibiting Wnt signaling.31 It accomplishes this by inhibiting all four activators
of the Wnt/β‐catenin pathway, including ΔNLRP6, FlagAx2, ΔN β‐catenin, and TCF4‐VP16.31
It is felt that LECT2 must be working at the levels of the TCF4 transcription factor
or below to be able to inhibit all four of these activators. Additionally, tumor necrosis
factor alpha, interferon‐γ, and adenosine monophosphate‐activated protein kinase all
seem to downregulate LECT2 expression in mice, but the exact mechanism remains unclear.2,
22, 23 We will discuss further molecular pathways in the sections below.
Current quantitation and analytical methods
Current studies have used several different methods of analyzing LECT2 in subjects.
Clinical studies on most species were performed using enzyme‐linked immunosorbent
assay (ELISA) plates.14, 53 ELISA plates using antibodies from across a wide spectrum
of species have been developed and are readily obtained from a number of biomedical
companies. Other studies have used Suppression Subtractive Hybridization, Northern
Blot, Western Blot, immunohistochemistry, and real‐time reverse‐transcriptase polymerase
chain reaction (PCR).9, 40 Although all of these methods are available for research
purposes, quantification is not yet readily available in the clinical setting at this
time and further studies are needed.
NORMAL FUNCTION AND CLINICAL SIGNIFICANCE
Cell cycle and development
Bovine chondromodulin‐II was identified before human LECT2 discovery and is structurally
very similar, as discussed before.45
In vitro, exposure to chondromodulin‐II stimulated the growth of rabbit growth‐plate
chondrocytes, mouse MC3T3‐E1 cells, and rat UMR‐106 osteoblastic cells.41, 47 This
stimulation of bone growth hints at a role in inducing cell division, proliferation,
and development, especially when considering that LECT2 is upregulated by the β‐catenin
/ WNT pathway. This pathway is involved in a wide range of functions, including cell
fate specification, cell proliferation, and cell migration.9 In fact, LECT2 is being
increasingly studied in the role of cellular development across the body. LECT knockout
mice have been found to have decreased development of axons in neurons as well as
altered expression of neutrophils that can affect neuronal development.21
Immune modulation and infection
Although there was evidence of neutrophil chemotaxis in vitro at the time of its discovery,
the full function of LECT2 in the immune system is not known.1, 9 Several studies
have attempted to clarify this but the results are nuanced and its function is not
easily classified as either pro‐inflammatory or anti‐inflammatory.
In mice, LECT2 has been found to activate macrophages in times of sepsis via the CD209a
receptor when placed in models of Escherichia coli sepsis, Pseudomonas aeruginosa
sepsis, and cecal ligation and puncture.10 In fact, mice that died at 24 h in the
Escherichia coli model had lower levels of circulating LECT2 (15.21 ± 1.03 ng/mL vs.
22.02 ± 1.22 ng/mL; P < 0.001) which may correlate with humans studies that we will
discuss below.10 In that same study, recombinant LECT2 administration at a concentration
of 30–135 ng/mg improved mouse survival from these models when compared with normal
saline.10, 48 Dang et al.14 demonstrated an increased lethality from staphylococcal
enterotoxin A in LECT2 knockout (LECT2‐KO) mice.14 More promisingly, exogenous LECT2
administration (5 μg/mouse at 0.5 h and 6 h) improved mouse survival from in those
same mice from <30% to ∼50% (P < 0.05).14 LECT2 can also upregulate macrophage gene
expression in certain species of fish, which again implies a conserved function across
species.12, 15
The role of LECT2 in sepsis in humans is also being investigated. A Japanese study
of 23 patients with sepsis admitted to the intensive care unit with confirmed infections
and 31 healthy volunteer patients found that on admission to the intensive care unit,
patients with sepsis initially had decreased circulating levels of LECT2 compared
with the healthy controls (5.3 ± 4.1 ng/mL vs. 19.7 ± 3.4 ng/mL; P < 0.0001) that
then increased by the time of discharge but was still less than the healthy controls.13
LECT2 was also found to be inversely and strongly correlated to C‐reactive protein,
a commonly used inflammatory marker. Patients who recovered had an increase in LECT2
values during their admission, whereas the one patient who died in the intensive care
unit had decreasing levels just prior to death. However, the study was not designed
to comment on correlation vs. causation. It was unable to determine if the low levels
of LECT2 contributed to disease severity or if it was simply downregulated in the
initial setting of sepsis.
Bone health
In humans, LECT2 has been identified as a human biomarker for the severity of osteoarthritis
(OA) by Ikeda et al.18 In that study, researchers obtained cartilage from six healthy
controls and 17 patients with OA. These samples were analyzed using isobaric tags
for relative and absolute quantitation and Western blot. LECT2 was found to be increased
in patients with OA as well as in patients >65 years of age with P < 0.05. This is
especially intriguing when taken in the context of a mouse model by Wu et al.20 in
2010. They found that mice with β‐catenin upregulation would convert normal articular
chondrocytes into arthritic chondrocytes leading to an OA phenotype by 5 months of
age, which is earlier compared with wild‐type (WT) mice.20 This study did not specifically
analyze LECT2; however, it is known that β‐catenin increases LECT2 expression and
that LECT2 in increased in human OA.9, 18
Given its effects on immune modulation and bone health, it is not surprising that
LECT2 has a role in auto‐immune or RA. Okumura et al.16 assessed LECT2‐KO mice compared
with WT mice with induction of arthritis by arthritogenic monoclonal antibody cocktails
at 6–7 weeks of age and then assessed 5 days later. These mice were assessed via hind
paw swelling, histology, ELISA, and RNA analysis. LECT2‐KO mice had worsening hind
paw thickness and characteristics of articular inflammation on histology (P < 0.05).
Certain chemokines and ILs (specifically IL‐1β and IL‐6) were found to be higher in
the hind paws of LECT2‐KO mice by 1.8‐fold and 2.8‐fold, respectively. A set of LECT2‐KO
mice were also given complementary LECT2 via hydrodynamic gene transfer after induction
of arthritis. These mice all had improvement in hind paw thickness and decreased concentrations
of IL‐1β, IL‐6, and other chemokines.
This work has been corroborated in cohort studies of Japanese and German populations.
In these studies, certain LECT2 polymorphisms, specifically Val58Ile, were associated
with worse cases of RA.19 Kameoka et al.19 obtained PCR studies of the LECT2 gene
in 101 Japanese volunteers. In this study, there was a clear association between the
genotypes 172AA (Ile/Ile) and 172GA (Val/Ile) and higher stage of RA (worsening disease
severity) compared with 172GG (Val/Val; P = 0.017). These findings were confirmed
by Graessler et al.,17 who found that in 204 patients with RA (using 81 patients with
OA and 116 patients with gout as controls), those carrying the 172AA (Ile/Ile) genotype
had worsening Larsen scores compared with those with 172GA (Val/Ile) and 172GG (Val/Val)
genotypes (96.8 vs. 69.5 vs. 54.8; P = 0.001). However, these studies did not focus
on the direct molecular causation of RA, but rather investigated the correlation of
polymorphisms to disease presence or severity.
Hepatology
There is a developing body of evidence that LECT2 can either reflect or impact liver
health, disease, and regeneration (also refer to below sections on metabolism, amyloidosis,
and oncology) in mice and humans. For example, LECT2‐KO mice have significantly more
severe liver injury following concanavalin A (Con A)‐induced hepatitis with an increased
hepatic natural killer T cells in their livers.11 Saito et al.11 were able to generate
these data by using targeting vectors to splice out LECT2 exons in mice. At baseline,
LECT2‐KO mice had increased intrahepatic levels of CD3int NK1.1+ cells compared with
the control group (26.1 ± 6.2% vs. 16.0 ± 4.6%; P < 0.01) as well as of CD4+ NK1.1+
cells (19.3 ± 4.8% vs. 11.1 ± 3.5%; P < 0.01). LECT2‐KO mice also had almost twice
the levels of Va14 natural killer T cells at baseline. The group then induced hepatitis
in WT and LECT2‐KO mice using 25 mg/kg Con A. At 5 h after Con A administration, LECT‐KO
mice had elevated serum alanine aminotransferase (ALT) and IL‐4 as well as hepatic
expression of FasL compared with WT mice. Livers from these mice were obtained at
5 h and examined histologically. Only the LECT2‐KO mice had focal degenerative change
and cell clusters of apoptotic cells.
Segawa et al.2 were able to identify a reduction in LECT2 expression in mice with
hepatic injury induced by Con A. The Con A (13 mg/kg) was administered i.v. to mice.
At 8 h after infusion, there was a decrease in LECT2 seen on Western blot with a sharp
increase in serum ALT and DNA fragmentation in the liver. This inverse relationship
reversed at 24 h and then went back to baseline between 48 and 96 h (P < 0.01). LECT2
expression was also inversely related to tumor necrosis factor alpha and interferon‐γ.
Several groups have also begun to apply this knowledge to humans. A Japanese study
by Sato et al.3 followed LECT2 serum levels in cases of living related donor liver
transplantation. They trended LECT2 levels using ELISA in five recipients and their
five donors and compared these values with other routine bloodwork. In both groups,
LECT2 initially decreased with a nadir at 3–12 h for the donors and 12–48 h in the
recipients, which is similar to the findings in mice by Segawa et al.2 Serum AST and
ALT were inversely related to LECT2 with a peak between 12 and 24 h in both donors
and recipients suggesting a role in liver regeneration. Interestingly, the serum LECT2
levels of donors were significantly higher than those of recipients on day 5 and 7
(9.5 +/‐ 5.9 ng/mL vs. 3.1 +/‐ 2.2 ng/mL; P = 0.04 and 9.3 +/‐ 3.8 ng/mL vs. 3.5 +/‐
1.1 ng/mL; P = 0.04).
The same group also followed six adult patients with acute liver failure who were
admitted to their hospital in 2002.6 The patients who died had significantly lower
peak serum LECT2 levels than those who survived (0.96 +/‐ 0.8 ng/mL vs. 12.9 +/‐ 4.3
ng/mL). Although these studies need to be repeated with larger numbers of patients,
they suggest a strong correlation with LECT2 and liver regeneration. These may be
impacted by the Wnt/β‐catenin pathway, which is related to liver regeneration.
LECT2 expression is directly regulated by the Wnt/β‐catenin pathway and it is important
to discuss hepatic functions of LECT2 in the context of the Wnt/β‐catenin pathway,
which we will discuss.9 Unfortunately, β‐catenin is an intracellular protein and is
unable to be measured without obtaining a liver biopsy. Because LECT2 is upregulated
after Wnt/β‐catenin activation and secreted into the bloodstream, it can be theorized
that LECT2 could be used as a reflection of Wnt/β‐catenin activation, liver regeneration,
or even as a potential therapeutic target of the Wnt/β‐catenin pathway. There are
ongoing investigations evaluating these possibilities and we will review some important
features of the Wnt/β‐catenin pathway below as future areas of possible research for
LECT2.
Mouse and human studies have described the correlation of liver regeneration and the
Wnt/β‐catenin pathway. Mouse models dosed with nonlethal 300 mg/kg of acetaminophen
showed extensive liver injury, but robust liver regeneration with several different
pathways activated, as outlined in a study by Apte et al.4 and Bhushan et al.5 These
pathways included IL‐6/STAT‐3, epidermal growth factor receptor / c‐Met / mitogen‐activated
protein kinase regeneration, and the Wnt/β‐catenin pathway.5 However, when dosed with
a lethal level of acetaminophen (600 mg/kg), mice had more extensive injury (higher
serum ALT levels and percentage necrosis areas on histology), less regeneration, and
decreased survival (P < 0.05).5 Interestingly, most regeneration pathways were still
activated in these lethally dosed mice, except for the Wnt/β‐catenin pathway, suggesting
a critical role in liver health. The study went further and bred mice with an overexpression
of a more stable β‐catenin protein and compared lethal and nonlethal APAP dosing to
WT mice. Overexpression of stable β‐catenin mice had improved serum ALT, percentage
necrosis areas, and perinuclear neutrophil antibodies (pANCA) protein expression in
hepatocytes suggesting regeneration.5
Knockout β‐catenin mice have also been studied for overall liver health. These mice
were found to have increased levels of hepatic apoptosis and decreased serum ascorbic
acid (vitamin C) levels by Nejak‐Bowen et al.7 This apoptosis was alleviated by ascorbic
acid administration to cultured hepatocytes. Another study of β‐catenin knockout mice
by Tan et al.8 showed an overall lower liver weight at 1 month of age that persisted
throughout adulthood. When these mice were subjected to partial hepatectomy they had
a slowed liver regeneration and increased apoptosis compared with WT mice.8 However,
regeneration did eventually occur indicating several redundant pathways in the liver.
These animal studies have provided a foundation of knowledge that is slowly being
applied to human studies as well. Retrospective liver samples were obtained by Apte
et al.4 of adult acetaminophen‐induced acute liver failure to assess β‐catenin activation.
Patients who survived had higher numbers of proliferating cell nuclear antigen staining
cells indicating cell division / regeneration.4 These same samples also stained highly
for intracellular β‐catenin.4 Samples from nonsurvivors showed decreased staining
for proliferating cell nuclear antigen and β‐catenin.
Metabolic syndrome
LECT2 is also implicated in nutritional and metabolic syndrome pathways. A study of
200 Japanese individuals by Lan et al.22 presenting for their yearly physical found
that there was a correlation between serum LECT2 levels and several aspects of metabolic
syndrome. There was a positive correlation on linear regression with body mass index,
waist circumference, homeostatic model assessment for insulin resistance, selenoprotein
P, hemoglobin A1c, and systolic blood pressure with statistical significance in humans.
That same report then compared LECT2‐KO and WT mice and found that LECT2 had a positive
correlation with markers of insulin resistance of skeletal muscle cells using models
of heat production, running endurance, intraperitoneal glucose administration, and
insulin tolerance tests. Interestingly, Lan et al.22 repeated a similar experiment
using LECT2‐KO and WT mice after being fed a high‐fat diet (HFD) to create an obesity
model. These obese mice still showed the same correlation with LECT2 and insulin resistance.
LECT2‐KO mice had decreased overall weight gain at 14 weeks (P < 0.001), decreased
blood glucose and insulin levels after meals (P < 0.05), and improved markers after
intraperitoneal glucose and insulin tolerance tests (P < 0.01).
Using these mouse models, Lan et al.22 was then able to identify two molecular pathways
that could lead to insulin resistance using Western blot and reverse transcribed RNA
with real time PCR analysis. The first pathway involves the phosphorylation of the
c‐JNK in C2C12 myocytes that has been implicated as a central player in obesity and
insulin resistance.22, 25, 26 Additionally, LECT2 was also found to decrease Akt phosphorylation,
which would then lead to the decreased translocation of glucose transporter 4.22,
27 The identification of these specific pathways could be an area of interest for
future targeted therapies of type 2 diabetes mellitus, a known consequence of insulin
resistance.
Insulin resistance is also a key factor in the development of nonalcoholic fatty liver
disease (NAFLD). Okumura et al.41 described an increase in the serum LECT2 levels
in patients with NAFLD compared with those patients without NAFLD in a cross‐sectional
study of 231 adult Japanese subjects (48.7 ± 13.6 ng/mL vs. 40.5 ± 12.8 ng/mL; P <
0.001). Again, this study found positive correlations on linear regression with LECT2
and body mass index, waist circumference, waist to hip ratio, and waist to height
ratio. They were also able to define cutoff values for LECT2 in the screening of obesity
(41.8 ng/mL in men, 45.0 ng/mL in women) and NAFLD (43.3 ng/mL for men, 46.4 ng/mL
for women) to help identify patients with these conditions. This suggests that LECT2
could be used as a biomarker in the setting of patients at risk for NAFLD.
Additional studies have identified molecular pathways for this process. Hwang et al.23
used HepG2 human HCC cell lines to study the effects of LECT2 on liver cells. They
wanted to assess its effects on molecular markers of fatty liver disease. Through
Western blot analysis, they found that LECT2 significantly increased phosphorylated
mammalian target of rapamycin and SREBP‐1 cleavage as well as JNK phosphorylation
via the CD209 receptor, which is similar to the activation of macrophages. In fact,
after LECT2 administration, these human cell lines were found to have increased lipid
accumulation on Oil Red O staining as well.
This may reflect the role of LECT2 in pathogenesis of NAFLD as LECT2 decreases insulin
sensitivity and could worsen the metabolic syndrome that contributes to the development
of NAFLD. However, as LECT2 plays a role in liver regeneration, it may be upregulated
as the liver attempts to recover from NAFLD leading to worsening insulin resistance
and further liver damage. This would need further clarified in future studies.
Recently, there have been studies examining LECT2 as a potential therapeutic target
for insulin resistance and NAFLD. Dipeptidyl peptidase‐4 inhibitors are a class of
medications used to treat insulin resistance via reducing cleavage of glucagon‐like
peptide‐1 and increasing its insulinotropic action.52 Hwang et al.23 used gemigliptin,
a dipeptidyl peptidase‐4 inhibitor, in mice fed an HFD that normally results in NAFLD
and followed their outcomes compared with mice fed an HFD alone in addition to the
HCC cell lines that were previously mentioned.23 They found that LECT2 expression
was inhibited by gemigliptin via increased adenosine monophosphate‐activated protein
kinase phosphorylation and JNK inhibitor‐dependent pathways.23 Additionally, mice
fed an HFD treated with gemigliptin had improved markers of disease for NAFLD and
insulin resistance compared with those fed an HFD alone. These included weight gain,
blood tumor necrosis factor alpha levels, lipid accumulation in the liver on histology,
and lower blood glucose levels after glucose tolerance testing.
Another aspect of metabolic syndrome that can lead to significant morbidity and mortality
is atherosclerosis. LECT2 has also been implicated in the development of atherosclerotic
blood vessels. This was again found to be regulated by the JNK phosphorylation and
activation via the CD209 receptor, which shares the JNK pathway with insulin resistance.24,
25, 26 Various doses of LECT2 were given to human umbilical vein endothelial cells
in this study by Hwang et al.23 Through Western blot and quantitative real‐time PCR,
this group was able to demonstrate induction of pro‐inflammatory molecules, such as
intercellular adhesion molecule 1, tumor necrosis factor alpha, monocyte chemo‐attractant
protein‐1, and IL‐1β that are known to lead to atherosclerosis. Given that LECT2 is
elevated in the setting of insulin resistance and is associated with NAFLD and atherosclerosis,
LECT2 has been proposed as a potential target for future medical therapies.
Amyloidosis
Amyloidosis is a rare yet serious disease in which the abnormal folding of proteins
lead to decreased proteolysis, the development of oligomers, and then deposition of
these oligomers into various organs. This leads to a variety of phenotypes based on
the specific proteins and organs involved. Within the last decade, a novel cause of
amyloidosis has been linked to the abnormal folding of the LECT2 protein. This was
first described in 2008 by Benson et al.34 in a patient with nephrotic syndrome of
initially unknown etiology. The disease has since been expanded to include hepatic,
splenic, adrenal glands, and pulmonary involvement and may represent as many as 25%
of hepatic cases.35, 39 Hepatic leukocyte chemotactic factor‐associated amyloidosis
most commonly presents as globular hepatic amyloid within the hepatic sinusoids and/or
portal tracts.38 Little is known about the natural history of this disease especially
how it is triggered and its prognosis. Data suggest that it is most common in patients
of Mexican ancestry and autopsy studies have shown that these amyloid deposits can
be found in the kidneys of up to 3.1% in patients of Hispanic descent.35, 36, 37 There
is no single disease causing mutation currently discovered; however, many patients
have been found to have a polymorphism of the G nucleotide in a nonsynonymous single
nucleotide polymorphism at position 172 of the LECT2 gene, again most commonly found
in patients from Latin America.36 This polymorphism, although common, is not sufficient
for disease progression and it is suspected that there is some yet to be identified
second hit to cause this disease.
Oncology
Given that uncontrolled inflammation and cell division can lead to malignancy, it
should come as no surprise that LECT2 has become a protein of interest in cancer pathogenesis.
Investigations using HCC and hepatoblastoma liver resections and RNA samples revealed
that LECT2 was upregulated in almost all hepatoblastoma (13/14 samples) and only a
subset of HCC (5/15 samples with and 6/33 samples without β‐catenin mutations).9 This
correlates well with the knowledge that the deregulation of the Wnt / β‐catenin pathway
is altered in >90% of hepatoblastoma and only 30–40% of HCC because LECT2 expression
is regulated by the Wnt/β‐catenin pathway.9, 32 Other studies show that LECT2 expression
can correlate with the stage of HCC. A study by Uchida et al.53 in 1999 used immunostaining
to analyze the expression of LECT2 in atypical hyperplasia (a premalignant lesion),
low‐grade malignant HCC, and advanced HCC.53 They found that as the HCC progressed
from atypical hyperplasia to advanced HCC, hepatocytes went from generally positive
for LECT2 in the cytoplasm of cells to generally negative staining and they provided
sample images from the immunostaining.53 This fact likely contributes to the variable
LECT2 expression in HCC seen in the Ovejero et al.9 study as lesions are found in
different stages in patients. Okabe et al.29 was also able to demonstrate LECT2 expression
by β‐catenin in an HCC mouse model and that mutations in β‐catenin led to increased
serum LECT2. Indeed, their β‐catenin knockout mice had 117‐fold decreased expression
of LECT2 compared with WT mice. When assessing tumor burden, their mice with histologic
tumors had increased LECT2 compared with those without (55.9 ± 19.9 ng/mL vs. 24.9
± 5.5 ng/mL; P < 0.01).
Interestingly, even though this upregulated gene expression is only present in a subset
of patients with HCC, there is still evidence that elevated levels of LECT2 can be
used as a biomarker of HCC in general regardless of β‐catenin mutation. Despite β‐catenin
mutations, Okabe et al.29 was able to observe that serum levels >50 ng/mL of LECT2
were able to identify human patients with HCC with a specificity of 96.1% and positive
predictive value of 97.0%. Given the potential role of LECT2 in the immune system,
NAFLD, and liver regeneration, in addition to HCC, the clinical utility of LECT2 may
be as a standalone test or as part of a battery of testing to personalize care. Mouse
models also suggest a utility of LECT2 as a hepatic biomarker for cancer. Although
β‐catenin is mutated and active in both colon and liver cancer and mice, only hepatic
malignancies resulted in LECT2 upregulation suggesting that LECT2 is specific for
hepatic malignancy in mice.9 Further mouse models have also demonstrated that LECT2
is a Wnt‐inhibitor and loss of LECT2 function may contribute to unregulated Wnt‐signaling,
which is found in intestinal adenoma formation as was demonstrated by Phesse et al.31
using mouse models of Mbd2 deficiency. All of this suggests that increased LECT2 may
be associated with hepatic malignancy but decreased or inhibited LECT2 may be associated
with intestinal malignancy.
Additional studies have mapped a direct molecular pathway for the inhibition of vascular
invasion in HCC by LECT2, which can correlate with survival. One study in particular
by Chen et al.28 was able to assess the effect of LECT2 in both mice and human subjects
as well as HCC cell lines. LECT2 was able to recruit protein tyrosine phosphatase
1B by binding to the HxGxD motif and decrease MET phosphorylation decreasing HCC's
ability for vascular invasion and metastasis. This was correlated with decreased vascular
invasion in human HCC (high LECT2 levels <20% vascular invasion, low LECT2 levels,
and 100% vascular invasion; P < 0.001) and improved the Kaplan‐Meier analysis for
survival. This may explain one of the changes that occurs as HCC progresses to its
advanced stages, loses LECT2 expression, and results in increased vascular invasion
and metastasis.
In mouse models of HCC development analyzed by Anson et al.,30 β‐catenin activation
was associated with release of both pro‐inflammatory and anti‐inflammatory mediators,
which creates a smoldering environment for HCC development. The main anti‐inflammatory
mediators seen by real time PCR included LECT2 and it was associated with invariant
natural killer T cells identified by flow cytometry and histology. The study group
then developed LECT2 and invariant natural killer T single and double knockout mice,
which developed β‐catenin‐activated HCC characterized by poorly controlled inflammation
leading to significant lung metastases and more than double the number of tumor nodules
seen in the controls.30 Interestingly, although β‐catenin activation generally seems
to be pro‐inflammatory and oncogenic, it can also signal a negative feedback cascade
using a somewhat anti‐inflammatory LECT2 signal that creates a more subtle inflammatory
process for the creation of HCC.
Last, LECT2 may have a role in predicting the outcomes of patients with nonhepatic
malignancies. LECT2 gene expression was found to be altered by smoking status.33 This,
in combination with CYP1A1 and CETN1, was found to help predict breast carcinoma recurrence
and survival among female smokers with median C‐index values of 0.8 and 0.73 for survival
and recurrence, respectively. Conversely, nonsmokers only had a median C‐index value
of 0.59.33
CONCLUSION AND FUTURE DIRECTIONS
These wide and varied research studies show how complex the role of LECT2 is in the
human body. It has many important functions in almost every system in the human body
from controlling and modulating the immune system, altering glucose metabolism, increasing
bone growth, controlling cell cycle and division, and likely many others yet to be
discovered. The clinical implications of changing levels of LECT2 in various disease
states are just as varied (Table
1).6, 13, 17, 18, 22, 28, 29, 33, 41, 42 Given the novelty of this gene and protein,
there are many areas that need further study and should be actively investigated.
Additionally, many of these pathophysiologic functions interact with each other, and
how they interplay with each other in the setting of different diseases needs to be
addressed.
In the liver, LECT2 is implicated in the development of metabolic syndrome. Increased
circulating levels of LECT2 are also associated with NAFLD. The development of insulin
resistance and NAFLD could have a common mediator with LECT2. Additionally, LECT2
may also reflect liver regeneration and studies explaining the pathway of both NAFLD
and liver regeneration through LECT2 should be clarified. Given the spectrum of liver
diseases associated with LECT2, it may be beneficial to analyze LECT2 as part of an
algorithm as opposed to an isolated test. For example, LECT2 could aid in NAFLD activity
score assessments for which patients would progress to liver cirrhosis. LECT2 could
also be used as a predictor for which patients with metabolic syndrome would develop
NAFLD or atherosclerosis. Last, there are preliminary investigations of using LECT2
as a therapeutic target in mice, but human studies, including which drug, dosing,
duration of therapy, and specific patient populations, still need to be performed.
It is also be important to note that hepatoblastoma and certain HCCs are associated
with different levels of LECT2 and would need to be carefully considered in any screening
guidelines. Further studies are needed in different hepatic malignancies to see how
specific and sensitive LECT2 is for particular cancers. Additionally, LECT2 expression
may have utility as a biomarker in breast cancer and could also be assessed as part
of a battery of tests for various malignancies.
There is also increasing awareness of the abnormal folding and build of LECT2 in hepatic,
renal, and pulmonary amyloidosis. Although a specific at‐risk polymorphism has been
identified with some phenotypic description, there is significantly more work to be
done in the fields of pathophysiology, genotype‐phenotype correlations, screening
tests, environmental influences, prevention, and treatment. The same can be said regarding
bone health and the role of LECT2 in arthritis syndrome.
Last, there may be several clinically applicable aspects of this protein in the setting
of immunology and infectious disease. However, before LECT2 can likely be used clinically
in humans, we need to understand how it modulates the immune system and through what
mechanisms of action. For example, it is known to activate macrophages in mice, but
there have been no conformational studies in human. In humans, levels of LECT2 are
associated with severity of sepsis, but the mechanism of action and whether this is
correlation or causation needs further study.
There are many areas of interest for the study of LECT2, but most research is still
in the beginnings of understanding the basics of this protein. Further research to
clarify the role of LECT2 across a multitude of diseases would allow LECT2 to have
an important future as a biomarker and therapeutic target (Table
2).2, 3, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 22, 23, 24, 28, 29, 30, 31,
33, 34, 35, 36, 37, 38, 39, 40, 42, 43