1,720,973 research outputs found
Transglutaminases: Expression in Kidney and relation to Kidney Fibrosis
No full textKidney fibrosis is regarded as a chronic wound response and tissue remodeling process consequent to a persistent
tissue damage. The fibrosis is characterized by excessive extracellular matrix accumulation, fibroblast proliferation
and chronic inflammation that leads to loss of tissue architecture and function. Transglutaminase-2 (TG2) is an
essential component of wound repair and overexpression of TG2 and/or excessive crosslinking by TG2 have been
notably linked to the pathogenesis of fibrosis in various organs. In this chapter, we discuss chronic kidney disease as
one of the most prevalent chronic diseases, whose characteristic trait is fibrosis. We attempt to review recent thinking
on the cellular and molecular causes of kidney fibrosis and review studies linking the role and enzymatic activity of
TG2 with the pathogenesis of kidney fibrosis. Additionally we discuss how TG2 represents a new treatment target,
which has catalyzed advances in the treatment of kidney disease
A crucial sequence for transglutaminase type 2 extracellular trafficking in renal tubular epithelial cells lies in its N-terminal -sandwich domain.
No full textTransglutaminase type 2 (TG2) catalyzes the formation of an
-(-glutamyl)-lysine isopeptide bond between adjacent peptides
or proteins including those of the extracellular matrix
(ECM). Elevated extracellular TG2 leads to accelerated ECM
deposition and reduced clearance that underlie tissue scarring
and fibrosis. The extracellular trafficking of TG2 is crucial to its
role in ECM homeostasis; however, the mechanism by which
TG2 escapes the cell is unknown as it has no signal leader peptide
and therefore cannot be transported classically. Understanding
TG2 transport may highlight novel mechanisms to
interfere with the extracellular function of TG2 as isoform-specific
TG2 inhibitors remain elusive. Mammalian expression vectors
were constructed containing domain deletions of TG2.
These were transfected into three kidney tubular epithelial cell
lines, and TG2 export was assessed to identify critical domains.
Point mutation was then used to highlight specific sequences
within the domain required for TG2 export. The removal of
-sandwich domain prevented all TG2 export. Mutations of
Asp94 and Asp97 within the N-terminal -sandwich domain
were identified as crucial for TG2 externalization. These form
part of a previously identified fibronectin binding domain
(88WTATVVDQQDCTLSLQLTT106). However, siRNA knockdown
of fibronectin failed to affect TG2 export. The sequence
88WTATVVDQQDCTLSLQLTT106 within the -sandwich
domain of TG2 is critical to its export in tubular epithelial cell
lines. The extracellular trafficking of TG2 is independent of
fibronectin
Fibronectin-tissue transglutaminase matrix rescues RGD-impaired cell adhesion through syndecan-4 and beta 1 integrin co-signaling
No full textHeterotropic association of tissue transglutaminase (TG2)
with extracellular matrix-associated fibronectin (FN) can
restore the adhesion of fibroblasts when the integrin-mediated
direct binding to FN is impaired using RGD-containing peptide.
We demonstrate that the compensatory effect of the TG-FN
complex in the presence of RGD-containing peptides is mediated
by TG2 binding to the heparan sulfate chains of the syndecan-
4 cell surface receptor. This binding mediates activation of
protein kinase C (PKC) and its subsequent interaction with
1 integrin since disruption of PKC binding to 1 integrins
with a cell-permeant competitive peptide inhibits cell adhesion
and the associated actin stress fiber formation. Cell signaling by
this process leads to the activation of focal adhesion kinase
and ERK1/2 mitogen-activated protein kinases. Fibroblasts
deficient in Raf-1 do not respond fully to the TG-FN complex
unless either the full-length kinase competent Raf-1 or the
kinase-inactive domain of Raf-1 is reintroduced, indicating
the involvement of the Raf-1 protein in the signaling mechanism.
We propose a model for a novel RGD-independent cell
adhesion process that could be important during tissue injury
and/or remodeling whereby TG-FN binding to syndecan-4
activates PKC leading to its association with 1 integrin,
reinforcement of actin-stress fiber organization, and MAPK
pathway activatio
Heparan sulphate proteoglycans are receptors for the cell-surface trafficking and biological activity of transglutaminase-2
No full textTransglutaminase type 2 (TG2) is both a protein cross-linking
enzyme and a cell adhesion molecule with an elusive unconventional
secretion pathway. In normal conditions, TG2-mediated
modification of the extracellular matrix modulates cell motility,
proliferation and tissue repair, but under continuous cell insult,
higher expression and elevated extracellular trafficking of TG2
contribute to the pathogenesis of tissue scarring. In search of
TG2 ligands that could contribute to its regulation, we characterized
the affinity of TG2 for heparan sulfate (HS) and heparin,
an analogue of the chains of HS proteoglycans (HSPGs). By
using heparin/HS solid-binding assays and surface plasmon resonance
we showed that purified TG2 has high affinity for heparin/
HS, comparable to that for fibronectin, and that cell-surface
TG2 interacts with heparin/HS.Wedemonstrated that cell-surface
TG2 directly associates with the HS chains of syndecan-4
without the mediation of fibronectin, which has affinity for both
syndecan-4 and TG2. Functional inhibition of the cell-surface
HS chains of wild-type and syndecan-4-null fibroblasts revealed
that the extracellular cross-linking activity of TG2 depends on
the HS of HSPG and that syndecan-4 plays a major but not
exclusive role. We found that heparin binding did not alter TG2
activity per se. Conversely, fibroblasts deprived of syndecan-4
were unable to effectively externalize TG2, resulting in its cytosolic
accumulation. We propose that the membrane trafficking
of TG2, and hence its extracellular activity, is linked to TG2
binding to cell-surface HSPG
Development of an effective Chronic Kidney Disease (CKD) model in C57BL/6 mice with histology relevant to human disease
No full textBackground: Genetically modified mice are used to investigate disease and assess potential interventions.
However, research into kidney fibrosis is hampered by a lack of models of chronic kidney
disease (CKD) in mice. Recently, aristolochic acid nephropathy (AAN), characterised by severe tubulointerstitial
fibrosis, has been identified as a cause of end stage kidney disease and proposed as a
model of CKD. Published studies have used various dosing regimens, species and strains, with variable
outcomes. Therefore, we aimed to develop a standardised protocol to develop tubulointerstitial
fibrosis using pure aristolochic acid I (AAI) in C57BL/6 mice. Methods: AAI dose optimisation was
performed by intraperitoneal injection of AAI at varying dose, frequency and duration. Kidney function
was assessed by serum creatinine. Fibrosis was quantified by hydroxyproline levels and Masson’s
Trichrome staining. Specific collagens were measured by immunofluorescent staining. Results:
Single doses of AAI of 1 10 mg/kg caused acute kidney failure and death. Lower doses of 2.5
mg/kg needed to be administrated more than weekly to cause significant fibrosis. 3 mg/kg once
every 3 days for 6 weeks followed by a disease development time of 6 weeks after AAI led to reduced
kidney weight and function. Substantial tubulointerstitial fibrosis occurred, with males more
severely affected. Increased deposition of collagen I, III and IV contributed to fibrosis, with collagen
III and IV higher in males. Conclusions: AAN can be induced in C57BL/6 mice. The regimen of 3 mg/
kg every 3 days for 6 weeks followed by 6 weeks of disease development time gives substantial tubulointerstitial
fibrosis with lesions similar to those in humans. Copyrigh
Expression of transglutaminase-2 isoforms in normal human tissues and cancer cell lines: dysregulation of alternative splicing in cancer
No full textThe multiple enzymatic activities and functions
of transglutaminase type 2 (TG2) may be attributed to
alternative TG2 molecules produced by differential splic-
ing of TG2 mRNA. Different RNA transcripts of the
human TG2 gene (TGM2) have been identified, but the
expression of TG2 multiple transcripts has never been
systematically addressed. We have confirmed and ratio-
nalized the main TG2 variants and developed a screening
assay for the detection of alternative splicing of TG2, based
on real-time reverse-transcription PCR. We have quantified
the multiple TG2 transcripts in a wide range of normal
tissues and in cancer cell lines from four different sites of
origin. Our data show a significant correlation in the
expression of canonical and alternative TG2 isoforms in
normal human tissue, but differences in alternative splicing
of TG2 in cancer cell lines, suggesting that in cancer cells
the alternative splicing of TG2 is a more active process
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