Mark D. Gorrell

3-Dimensional imaging of collagen using second harmonic generation

Collagen is the most important structural protein of the animal body. Its unique triple-helix structure and extremely high level of crystallinity make it exceptionally efficient in generating the second harmonic of incident light, and we show here how this leads to a novel mode of microscopy of immediate practical significance in medicine and biology. In particular, it provides sensitive and high-resolution information on collagen distribution, discriminates between type I and type III collagen, and allows both a greater understanding of and a sensitive test for cirrhosis of the liver. Future research applications could include wound healing and hereditary collagen diseases such as osteogenesis imperfecta.

CD26: A Multifunctional Integral Membrane and Secreted Protein of Activated Lymphocytes

CD26 has proved interesting in the fields of immunology, endocrinology, cancer biology and nutrition owing to its ubiquitous and unusual enzyme activity. This dipeptidyl aminopeptidase (DPP IV) activity generally inactivates but sometimes alters or enhances the biological activities of its peptide substrates, which include several chemokines. CD26 costimulates both the CD3 and the CD2 dependent T‐cell activation and tyrosine phosphorylation of TCR/CD3 signal transduction pathway proteins. CD26 in vivo has integral membrane protein and soluble forms. Soluble CD26 is at significant levels in serum, these levels alter in many diseases and soluble CD26 can modulate in vitro T‐cell proliferation. CD26, being an adenosine deaminase binding protein (ADAbp), functions as a receptor for ADA on lymphocytes. The focus of this review is the structure and function of CD26 and the influence of its ligand binding activity on T‐cell proliferation and the T cell costimulatory activity of CD26.

Dipeptidyl peptidase IV and related enzymes in cell biology and liver disorders

DP (dipeptidyl peptidase) IV is the archetypal member of its six-member gene family. Four members of this family, DPIV, FAP (fibroblast activation protein), DP8 and DP9, have a rare substrate specificity, hydrolysis of a prolyl bond two residues from the N-terminus. The ubiquitous DPIV glycoprotein has proved interesting in the fields of immunology, endocrinology, haematology and endothelial cell and cancer biology and DPIV has become a novel target for Type II diabetes therapy. The crystal structure shows that the soluble form of DPIV comprises two domains, an alpha/beta-hydrolase domain and an eight-blade beta-propeller domain. The propeller domain contains the ADA (adenosine deaminase) binding site, a dimerization site, antibody epitopes and two openings for substrate access to the internal active site. FAP is structurally very similar to DPIV, but FAP protein expression is largely confined to diseased and damaged tissue, notably the tissue remodelling interface in chronically injured liver. DPIV has a variety of peptide substrates, the best studied being GLP-1 (glucagon-like peptide-1), NPY (neuropeptide Y) and CXCL12. The DPIV family has roles in bone marrow mobilization. The functional interactions of DPIV and FAP with extracellular matrix confer roles for these proteins in cancer biology. DP8 and DP9 are widely distributed and indirectly implicated in immune function. The DPL (DP-like) glycoproteins that lack peptidase activity, DPL1 and DPL2, are brain-expressed potassium channel modulators. Thus the six members of the DPIV gene family exhibit diverse biological roles.

Insights into the Pathobiology of Hepatitis C Virus-Associated Cirrhosis: Analysis of Intrahepatic Differential Gene Expression

The pathogenesis of hepatitis C virus (HCV)-associated liver injury involves many genes from multiple pathogenic pathways. cDNA array analysis, which examines the expression of many genes simultaneously, was used to achieve new insights into HCV liver injury. Membrane-based cDNA arrays of 874 genes compared HCV-associated cirrhosis with autoimmune hepatitis-associated cirrhosis as an inflammatory and cirrhotic control, and with nondiseased liver tissue. Array analysis identified many differentially expressed genes that are important in inflammation, fibrosis, proliferation, signaling, apoptosis, and oxidative stress. Genes up-regulated in HCV-associated cirrhosis were predominantly associated with a Th1 immune response, fibrosis, cellular proliferation, and apoptosis. Novel observations of differential gene expression included increased expression of secreted apoptosis-related protein 3, a Wnt pathway gene possibly involved in cellular apoptosis. EMMPRIN (CD147) and discoidin domain receptor 1 (CD167) were also shown to be increased and are likely to play a role in liver fibrosis. Real-time quantitative reverse transcriptase-polymerase chain reaction confirmed the increased expression of 15 genes. The comparison of HCV cirrhosis with autoimmune hepatitis cirrhosis showed a marked difference in the apoptosis-associated gene profile with HCV cirrhosis characterized by increased proapoptotic gene expression whereas autoimmune hepatitis was characterized by increased expression of both antiapoptotic and proapoptotic genes. Furthermore, expression of beta-catenin and the fibrosis-associated protein EMMPRIN were localized by immunohistochemistry to the plasma membranes of hepatocytes and biliary epithelium. In conclusion, HCV-associated cirrhosis was characterized by a proinflammatory, profibrotic, and proapoptotic gene expression profile.

Inhibitor selectivity in the clinical application of dipeptidyl peptidase-4 inhibition

DPP-4 (dipeptidyl peptidase-4) degrades the incretin hormones GLP-1 (glucagon-like peptide-1) and GIP (gastric inhibitory polypeptide), decreasing their stimulatory effects on beta-cell insulin secretion. In patients with Type 2 diabetes, meal-related GLP-1 secretion is reduced. DPP-4 inhibitors (alogliptin, saxagliptin, sitagliptin and vildagliptin) correct the GLP-1 deficiency by blocking this degradation, prolonging the incretin effect and enhancing glucose homoeostasis. DPP-4 is a member of a family of ubiquitous atypical serine proteases with many physiological functions beyond incretin degradation, including effects on the endocrine and immune systems. The role of DPP-4 on the immune system relates to its extra-enzymatic activities. The intracytosolic enzymes DPP-8 and DPP-9 are recently discovered DPP-4 family members. Although specific functions of DPP-8 and DPP-9 are unclear, a potential for adverse effects associated with DPP-8 and DPP-9 inhibition by non-selective DPP inhibitors has been posed based on a single adverse preclinical study. However, the preponderance of data suggests that such DPP-8 and DPP-9 enzyme inhibition is probably without clinical consequence. This review examines the structure and function of the DPP-4 family, associated DPP-4 inhibitor selectivity and the implications of DPP-4 inhibition in the treatment of Type 2 diabetes.

Identification of novel molecules and pathogenic pathways in primary biliary cirrhosis: cDNA array analysis of intrahepatic differential gene expression.

BACKGROUND Primary biliary cirrhosis (PBC) is an autoimmune disease in which the pathogenesis of progressive liver injury is poorly understood. AIM To provide novel insights into the pathogenesis of PBC related liver injury using cDNA array analysis, which simultaneously examines expression of many genes. METHODS Utilising cDNA arrays of 874 genes, PBC was compared with primary sclerosing cholangitis (PSC) associated cirrhosis and non-diseased liver. Differential expression of 10 genes was confirmed by real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS Array analysis identified many differentially expressed genes that are important in inflammation, fibrosis, proliferation, signalling, apoptosis, and oxidative stress. PBC was associated with increased expression of both Th1 and Th2 type molecules of the immune response. Fibrosis related gene expression featured upregulation of connective tissue growth factor and transforming growth factor beta3. Many more apoptosis associated molecules exhibited increased expression, consistent with apoptosis being a more active and regulated process, in PSC associated cirrhosis than in PBC. Increased expression of many genes of the Wnt and notch pathways implicated these highly conserved and linked pathways in PBC pathogenesis. The observed increases in expression of c-jun, c-myc, and c-fos related antigen 1 are consistent with increased Wnt pathway activity in PBC. Differential expression of four components of the Wnt pathway, Wnt-5a, Wnt-13, FRITZ, and beta-catenin, was confirmed by quantitative RT-PCR. CONCLUSION Many genes implicated in intrahepatic inflammation, fibrosis, and regeneration were upregulated in PBC cirrhosis. In particular, increased expression of a number of Drosophila homologues was seen in PBC.

The dipeptidyl peptidase IV family in cancer and cell biology

Of the 600+ known proteases identified to date in mammals, a significant percentage is involved or implicated in pathogenic and cancer processes. The dipeptidyl peptidase IV (DPIV) gene family, comprising four enzyme members [DPIV (EC 3.4.14.5), fibroblast activation protein, DP8 and DP9] and two nonenzyme members [DP6 (DPL1) and DP10 (DPL2)], are interesting in this regard because of their multiple diverse functions, varying patterns of distribution/localization and subtle, but significant, differences in structure/substrate recognition. In addition, their engagement in cell biological processes involves both enzymatic and nonenzymatic capabilities. This article examines, in detail, our current understanding of the biological involvement of this unique enzyme family and their overall potential as therapeutic targets.

Fibroblast activation protein increases apoptosis, cell adhesion, and migration by the LX‐2 human stellate cell line

Injury and repair in chronic liver disease involve cell adhesion, migration, apoptosis, proliferation, and a wound healing response. In liver, fibroblast activation protein (FAP) has both collagenase and dipeptidyl peptidase IV (DPIV) activities and is expressed only by activated hepatic stellate cells (HSC) and myofibroblasts, which produce and degrade extracellular matrix (ECM). FAP was colocalized with collagen fibers, fibronectin, and collagen type I in human liver. FAP function was examined in vitro by expressing green fluorescent protein FAP fusion protein in cell lines cultured on collagen‐I, fibronectin, and Matrigel. Glutamates at 203 and 204 as well as serine624 of FAP were essential for peptidase activity. Human embryonic kidney 293T cells overexpressing FAP showed reduced adhesion and migration. FAP overexpression in the human HSC line LX‐2 caused increased cell adhesion and migration on ECM proteins as well as invasion across transwells in the absence or presence of transforming growth factor beta‐1. FAP overexpression enhanced staurosporine streptomyces–stimulated apoptosis in both cell lines. Interestingly, the enzyme activity of FAP was not required for these functions. Overexpressing FAP increased the expression of matrix metalloproteinase‐2 and CD44 and reduced integrin‐β1 expression in 293T cells, suggesting potential pathways of FAP‐mediated impairment of cell adhesion and migration in this epithelial cell line. In conclusion, these findings further support a pro‐fibrogenic role for FAP by indicating that, in addition to its enzymatic functions, FAP has important nonenzymatic functions that in chronic liver injury may facilitate tissue remodeling through FAP‐mediated enhancement of HSC cell adhesion, migration, and apoptosis. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;42:935–945.)

Two highly conserved glutamic acid residues in the predicted β propeller domain of dipeptidyl peptidase IV are required for its enzyme activity

Dipeptidyl peptidase IV (DPP IV) is a member of the prolyl oligopeptidase family and modifies the biological activities of certain chemokines and neuropeptides by cleaving their N‐terminal dipeptides. This paper reports the identification and possible significance of a novel conserved sequence motif Asp‐Trp‐(Val/Ile/Leu)‐Tyr‐Glu‐Glu‐Glu (DW(V/I/L)YEEE) in the predicted β propeller domain of the DPP IV‐like gene family. Single amino acid point mutations in this motif identified two glutamates, at positions 205 and 206, as essential for the enzyme activity of human DPP IV. This observation suggests a novel role in proteolysis for residues of DPP IV distant from the Ser‐Asp‐His catalytic triad.

Stromal cell-derived factors 1α and 1β, inflammatory protein-10 and interferon-inducible T cell chemo-attractant are novel substrates of dipeptidyl peptidase 8

N‐terminal truncation of chemokines by proteases including dipeptidyl peptidase (DP) IV significantly alters their biological activity; generally ablating cognate G‐protein coupled receptor engagement and often generating potent receptor antagonists. DP8 is a recently recognised member of the prolyl oligopeptidase gene family that includes DPIV. Since DPIV is known to process chemokines we surveyed 27 chemokines for cleavage by DP8. We report DP8 cleavage of the N‐terminal two residues of IP10 (CXCL10), ITAC (CXCL11) and SDF‐1 (CXCL12). This has implications for DP8 substrate specificity. Chemokine cleavage and inactivation may occur in vivo upon cell lysis and release of DP8 or in the inactivation of internalized chemokine/receptor complexes.