Dipeptidyl Peptidase IV Enzyme Research Articles

Dipeptidyl peptidase IV (DPPIV), a candidate tumor suppressor gene in melanomas is silenced by promoter methylation

Dipeptidyl peptidase IV (DPPIV), a serine protease is expressed by normal melanocytes but not by melanomas, the malignant counterpart. DPPIV is encoded by a gene that contains a 5 CpG island spanning a transcriptional regulatory region. Previously we have demonstrated that DPPIV abrogates growth factor independence and functions as a tumor suppressor gene in melanomas. In this study we show that loss of DPPIV occurs at RNA level and demethylating agent, 5-aza-2'-deoxycytidine (5-AZA-Cdr) treatment of DPPIV negative melanoma cell lines results in increase of DPPIV mRNA, protein, and enzyme activities. By using sodium bisulfite genomic DNA modifications, PCR, and sequencing we confirmed that DPPIV gene promoter is methylated in eight out of ten melanoma cell lines tested. Further more, 5-AZA-Cdr induced increases in DPPIV levels correlated with growth inhibition and apoptosis in melanoma cells. All together these findings suggest that frequent downregulation of DPPIV expression in melanoma can be attributed, in large part, to aberrant promoter hypermethylation and this loss of DPPIV may be a critical event contributing to melanoma development.

Cholangiocyte marker-positive and -negative fetal liver cells differ significantly in their ability to regenerate the livers of adult rats exposed to retrorsine

We have used monoclonal antibodies against cell-surface developmental epitopes in combination with micromagnetic beads to isolate phenotypically defined subpopulations of cholangiocyte marker-positive fetal liver epithelial cells (CMP-FLEC). Differentiation potential was evaluated by injecting cell isolates from dipeptidyl peptidase IV (DPPIV) positive (DPPIV+) Fischer donor rats into the spleen of partially hepatectomized, DPPIV negative (DPPIV-) Fischer host rats exposed to retrorsine. At various time points, liver tissue was harvested and cells in DPPIV+ colonies were phenotyped by immunofluorescence and histochemical protocols. Functional differentiation and liver replacement were determined by comparing donor and host hepatocyte protein expression patterns and DPPIV enzyme activity in extracts from livers of host rats receiving CMP-FLEC. Our results showed that bipotentiality was retained during differentiation and maturation of CMP-FLEC, indicating that the acquisition of ductal morphology and phenotype were not indicative of lineage commitment. CMP-FLEC transplanted into the adult rat liver lost ductal and gained hepatocyte markers, and acquired protein expression patterns in 2D gels with a close similarity (>75% spot match) to host hepatocytes but differing significantly from the transplanted CMP-FLEC cell isolate (<25% spot match). The average size of donor hepatocyte colonies increased with time so that by 1 year, up to 70% of the host rat liver was replaced by CMP-FLEC derived DPPIV+ hepatocytes. Depletion of CMP-FLEC from fetal liver isolates resulted in a marked decrease in adult liver colonization, suggesting that a high percentage of the hepatocyte colonies in animals receiving total fetal liver isolates are derived from CMP-FLEC.

Sitagliptin: A Dipeptidyl Peptidase IV Inhibitor for the Treatment of Type 2 Diabetes

To review the pharmacology, pharmacokinetics, safety, and efficacy of sitagliptin, a dipeptidyl peptidase IV (DPP-IV) inhibitor in the management of type 2 diabetes mellitus. A MEDLINE search (1966-February 2006) was conducted for English-language articles using the terms dipeptidyl peptidase IV inhibitor, incretin, MK-0431, and sitagliptin. Abstracts from the American Diabetes Association annual meetings in 2004 and 2005 were included as sources of data. Articles pertaining to the pharmacology of sitagliptin, its pharmacokinetics, safety and efficacy were reviewed. Sitagliptin is a potent, competitive, reversible inhibitor of the DPP-IV enzyme. It is eliminated renally, with a terminal half-life of 11.8-14.4 hours. In Phase II clinical trials, sitagliptin was found to be superior to placebo for the treatment of type 2 diabetes mellitus. Results of a small trial comparing sitagliptin with glipizide indicate that both treatments are comparable. The efficacy of sitagliptin has also been demonstrated when used as adjunctive therapy with metformin. Few adverse effects have been reported. Weight gain and hypoglycemia have not been seen with sitagliptin therapy. Based on its unique mechanism of action, sitagliptin will provide practitioners with an additional tool in the treatment of diabetes. Review of the literature to date implies sitagliptin may be effective as monotherapy in type 2 diabetes. In addition, existing evidence supports the use of sitagliptin as adjunct therapy to sulfonylureas and metformin. Another advantage of sitagliptin use is that it appears to be free from the adverse effects of weight gain and hypoglycemia that are associated with currently available treatments.

Mutant HNF-1α and mutant HNF-1β identified in MODY3 and MODY5 downregulate DPP-IV gene expression in Caco-2 cells

Dipeptidylpeptidase IV (DPP-IV) is a well-documented drug target for the treatment of type 2 diabetes. Hepatocyte nuclear factors (HNF)-1α and HNF-1β, known as the causal genes of MODY3 and MODY5, respectively, have been reported to be involved in regulation of DPP-IV gene expression. But, it is not completely clear (i) that they play roles in regulation of DPP-IV gene expression, and (ii) whether DPP-IV gene activity is changed by mutant HNF-1α and mutant HNF-1β in MODY3 and MODY5. To explore these questions, we investigated transactivation effects of wild HNF-1α and 13 mutant HNF-1α, as well as wild HNF-1β and 2 mutant HNF-1β, on DPP-IV promoter luciferase gene in Caco-2 cells by means of a transient experiment. Both wild HNF-1α and wild HNF-1β significantly transactivated DPP-IV promoter, but mutant HNF-1α and mutant HNF-1β exhibited low transactivation activity. Moreover, to study whether mutant HNF-1α and mutant HNF-1β change endogenous DPP-IV enzyme activity, we produced four stable cell lines from Caco-2 cells, in which wild HNF-1α or wild HNF-1β, or else respective dominant-negative mutant HNF-1αT539fsdelC or dominant-negative mutant HNF-1βR177X, was stably expressed. We found that DPP-IV gene expression and enzyme activity were significantly increased in wild HNF-1α cells and wild HNF-1β cells, whereas they decreased in HNF-1αT539fsdelC cells and HNF-1βR177X cells, compared with DPP-IV gene expression and enzyme activity in Caco-2 cells. These results suggest that both wild HNF-1α and wild HNF-1β have a stimulatory effect on DPP-IV gene expression, but that mutant HNF-1α and mutant HNF-1β attenuate the stimulatory effect.

Identification and partial characterization of the enzyme of omega: one of five putative DPP IV genes in Drosophila melanogaster.

The omega (ome) gene product is a modifier of larval cuticle protein 5 and its alleles (and duplicates) in the third instar of Drosophila melanogaster. Using deletion mapping the locus mapped to 70F-71A on the left arm of chromosome 3. A homozygote null mutant (ome 1) shows a pleiotropic phenotype that affected the size, developmental time of the flies, and the fertility (or perhaps the behavior) of homozygous mutant males. The omega gene was verified as producing a dipeptidyl peptidase IV (DPPIV) by genetic analysis, substrate specificity and pH optimum. The identity of the gene was confirmed as CG32145 (cytology 70F4) in the Celera Database (Berkeley Drosophila Genome Project), which is consistent with its deletion map position. The genomic structure of the gene is described and the decrease in DPPIV activity in the mutant ome1 is shown to be due to the gene CG32145 (omega). The D. melanogaster omega DPPIV enzyme was partially purified and characterized. The exons of the ome1 mutant were sequenced and a base substitution mutation in exon 4 was identified that would yield a truncated protein caused by a stop codon. A preliminary study of the compartmentalization of the omega DPPIV enzyme in several organs is also reported.

CD26/Dipeptidyl Peptidase IV (DPPIV) Regulates p38 Phosphorylation and Topoisomerase II Alpha Expression in the B-Lymphoma Line Jiyoye, Associated with Enhanced In Vitro and In Vivo Sensitivity to Doxorubicin.

CD26 is a 110 kDa surface glycoprotein with diverse functional properties, including having a key role in normal T-cell biology, being a marker of aggressive disease for selected T-cell malignancies and being involved in the development of certain cancers. Its extracellular domain encodes a membrane-associated dipeptidyl peptidase IV (DPPIV) activity capable of processing biological factors to alter their functional profiles. We have shown previously that expression of CD26 on the T cell line Jurkat is associated with increased topoisomerase II alpha level and in vitro sensitivity to topoisomerase II inhibitors (Aytac U et al. Cancer Res 61:7204, 2001; Aytac U et al. Br J Cancer 88:455, 2003; Sato K et al. Br J Cancer 89:1366, 2003). We now show that CD26 expression, particularly its DPPIV enzyme activity, on the B-lymphoma line Jiyoye results in increased topoisomerase II alpha level and in vitro sensitivity to doxorubicin-induced apoptosis. Examining the molecular mechanisms involved in CD26-associated signaling, our present findings also indicate that CD26/DPPIV expression on Jiyoye cells is associated with increased phosphorylation of p38 and its upstream regulators MKK3/6 and ASK1. Importantly, inhibition of p38 phosphorylation decreases topoisomerase II alpha expression, suggesting a role for p38 in the regulation of topoisomerase II alpha. Finally, studies using a SCID mouse xenograft model with CD26 Jiyoye transfectants show that CD26 expression is associated with enhanced survival following treatment with low doses of doxorubicin. In particular, treatment with low-dose doxorubicin of SCID mice injected with CD26-negative parental Jiyoye cells does not lead to a statistically significant survival advantage over those treated with saline. On the other hand, SCID mice injected with CD26 Jiyoye transfectants show significantly greater survival when treated with low-dose doxorubicin than with saline alone, indicating that CD26 presence renders tumor cells more sensitive to doxorubicin in an in vivo model. Our data thus characterize the biochemical linkage among CD26 and other key intracellular molecules, while demonstrating that CD26 may have a role in tumor sensitivity to antineoplastic agents targeting topoisomerase II alpha. In addition, our work suggests that CD26/DPPIV may be an appropriate target for therapy for selected hematological malignancies of both B- and T-cell lineages.

Adenosine Down-Regulates the Surface Expression of Dipeptidyl Peptidase IV on HT-29 Human Colorectal Carcinoma Cells: Implications for Cancer Cell Behavior

Dipeptidyl peptidase IV (DPPIV) is a multifunctional cell-surface protein that, as well as having dipeptidase activity, is the major binding protein for adenosine deaminase (ADA) and also binds extracellular matrix proteins such as fibronectin and collagen. It typically reduces the activity of chemokines and other peptide mediators as a result of its enzymatic activity. DPPIV is aberrantly expressed in many cancers, and decreased expression has been linked to increases in invasion and metastasis. We asked whether adenosine, a purine nucleoside that is present at increased levels in the hypoxic tumor microenvironment, might affect the expression of DPPIV at the cell surface. Treatment with a single dose of adenosine produced an initial transient (1 to 4 hours) modest (approximately 10%) increase in DPPIV, followed by a more profound (approximately 40%) depression of DPPIV protein expression at the surface of HT-29 human colon carcinoma cells, with a maximal decline being reached after 48 hours, and persisting for at least a week with daily exposure to adenosine. This down-regulation ofDPPIV occurred at adenosine concentrations comparable to those present within the extracellular fluid of colorectal tumors growing in vivo, and was not elicited by inosine or guanosine. Neither cellular uptake of adenosine nor its phosphorylation was necessary for the down-regulation of DPPIV. The decrease in DPPIV protein at the cell surface was paralleled by decreases in DPPIV enzyme activity, binding of ADA, and the ability of the cells to bind to and migrate on cellular fibronectin. Adenosine, at concentrations that exist within solid tumors, therefore acts at the surface of colorectal carcinoma cells to decrease levels and activities of DPPIV. This down-regulation of DPPIV may increase the sensitivity of cancer cells to the tumor-promoting effects of adenosine and their response to chemokines and the extracellular matrix, facilitating their expansion and metastasis.

Dipeptidyl peptidase IV (DPPIV) enzyme activity on immature T-cell line R1.1 is down-regulated by dynorphin-A (1–17) as a non-substrate inhibitor

In this study we examined surface expression of CD26 and the corresponding enzyme activity of dipeptidyl peptidase IV (DPPIV) on the cells of immature murine T-cell line, R1.1. The data obtained have shown that R1.1 cells express high density of surface CD26 as compared to normal thymus cells. This was associated with strong enzyme activity, which, based on substrates and inhibitor specificity, corresponded to DPPIV. The DPPIV enzyme activity of R1.1 cells was 10 times stronger than that found on normal murine thymus cells (V max = 39 μmol/min/10 6 cells, vs 3.7 μmol/min/10 6 cells, respectively). Upon activation with anti-CD3, up-regulation of both membrane CD26, as well as of DPPIV enzyme activity on R1.1 cells were observed. The finding of strong DPPIV on R1.1 cells makes them suitable model for testing putative substrates/inhibitors of the enzyme in its natural microenvironment. Since in addition to strong DPPIV, R1.1 cells also express kappa opioid receptors (KOR) [European Journal of Pharmacology 227 (1992) 257], we tested the effect of dynorphin-A (1–17), an endogenous opioid peptide with KOR selectivity, on DPPIV of R1.1 cells. Dynorphin-A (1–17) down-regulated DPPIV in a dose-dependent manner, with the potency similar to that of substance P, a known natural DPPIV substrate [Journal of Pharmacology and Experimental Therapeutics 260 (1992) 1257]. DPPIV down-regulation was resistant to bestatin and thiorphan, the inhibitors of two cell surface peptidases (APN and NEP, respectively) with potential of dynorphin-A (1–17) degradation, suggesting that the mechanism underlying the observed effect does not involve degradative products of dynorphin-A (1–17). DPPIV down-regulation was also resistent to KOR antagonist, NBI, suggesting that the mechanism underlying the observed phenomenon involves neither cointernalization of KOR and DPPIV. Collectively, cells of immature T cell line, R1.1 exert strong DPPIV enzyme activity, which could be down-regulated in the presence of dynorphin-A (1–17) by mechanism that presumably includes non-substrate inhibition. By down-regulating DPPIV, dynorphin-A (1–17) may indirectly affect activity and/or specificity of natural substrates of DPPIV, such as substance P, RANTES, and endomorphins.

G1/S cell cycle arrest provoked in human T cells by antibody to CD26.

CD26 is T cell costimulatory molecule with dipeptidyl peptidase IV (DPPIV) enzyme activity located in its extracellular region. The expression of CD26 is enhanced after activation of T cells, while it is preferentially expressed on a subset of CD4+ memory T cells in the resting state. In this paper, we demonstrate that binding of the soluble anti-CD26 monoclonal antibody (mAb) 1F7 inhibits human T-cell growth and proliferation in both CD26-transfected Jurkat T-cell lines and human T-cell clones by inducing G1/S arrest, which is associated with enhancement of p21Cip1 expression. This effect depends on the DPPIV enzyme activity of the CD26 molecule. Moreover, we show that expression of p21Cip1 after treatment with the anti-CD26 mAb 1F7 appears to be induced through activation of extracellular signal-regulated kinase (ERK) pathway. These data thus suggest that anti-CD26 treatment may have potential use in the clinical setting involving activated T cell dysregulation, including autoimmune disorders and graft-vs.-host disease.

CD26/DPPIV signal transduction function, but not proteolytic activity, is directly related to its expression level on human Th1 and Th2 cell lines as detected with living cell cytochemistry.

CD26/DPPIV is a cell surface glycoprotein that functions both in signal transduction and as a proteolytic enzyme, dipeptidyl peptidase IV (DPPIV). To investigate how two separate functions of one molecule are regulated, we analyzed CD26 protein expression and DPPIV enzyme activity on living human T-helper 1 (Th1) and Th2 cells that express different levels of CD26/DPPIV. DPPIV activity was specifically determined with the synthetic fluorogenic substrate ala-pro-cresyl violet and CD26 protein expression was demonstrated with an FITC-conjugated CD26-specific antibody. Fluorescence of liberated cresyl violet (red) and FITC (green) was detected simultaneously on living T-cells using flow cytometry and spectrofluorometry. Th1 cells expressed three- to sixfold more CD26 protein than Th2 cells. The signal transduction function of the CD26/DPPIV complex, tested by measuring its co-stimulatory potential for proliferation, was directly related to the amount of CD26 protein at the cell surface. However, DPPIV activity was similar in both cell populations at physiological substrate concentrations because of differences in K(m) and V(max) values of DPPIV on Th1 and Th2 cells. Western blotting and zymography of Th1 and Th2 whole-cell lysates demonstrated similar patterns. This study shows that two functions of one molecule can be controlled differentially.

Increased Expression of Dipeptidyl Peptidase IV in Human Mesothelial Cells by Malignant Ascites from Ovarian Carcinoma Patients

Cell surface aminopeptidases play an important role in biological processes through degradation of small peptides. There are many bioactive peptides in ascites and these peptides are involved in carcinoma cell dissemination and infiltration. In human mesothelial cells dipeptidyl peptidase IV (DPPIV) shows the highest expression mostly in four cell surface aminopeptidases: aminopeptidase A, neutral endopeptidase 24-11, aminopeptidase N and DPPIV. Since mesothelial cells are always in contact with ascites, we examined the influence of malignant ascites on DPPIV. DPPIV enzyme activity in mesothelial cells was enhanced by the addition of ascites obtained from ovarian carcinoma patients in a time- and concentration-dependent manner, and flow cytometry and immunocytochemistry also revealed an increased expression of DPPIV on the cell surface of mesothelial cells. The <3-kD fraction of malignant ascites increased the DPPIV enzyme activity to the same level as the total ascites. Northern hybridization demonstrated that DPPIV mRNA was increased 3-fold by the addition of the <3-kD malignant ascites. In conclusion, DPPIV is highly expressed in human mesothelial cells and was regulated by ascites.

Soluble CD26/dipeptidyl peptidase IV enhances transendothelial migration via its interaction with mannose 6-phosphate/insulin-like growth factor II receptor

CD26 is a T cell surface molecule with dipeptidyl peptidase IV (DPPIV) enzyme activity in its extracellular region. In addition to its membrane form, CD26 exists in plasma as a soluble form (sCD26), which is the extracellular domain of the molecule thought to be cleaved from the cell surface. In this paper, we demonstrate that sCD26 mediates enhanced transendothelial T cell migration, an effect that requires its intrinsic DPPIV enzyme activity. We also show that sCD26 directly targets endothelial cells and that mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR) on the endothelial cell surface acts as a receptor for sCD26. Our findings therefore suggest that sCD26 influences T cell migration through its interaction with M6P/IGFIIR.

Association of Na+-H+ Exchanger Isoform NHE3 and Dipeptidyl Peptidase IV in the Renal Proximal Tubule

In an attempt to identify proteins that assemble with the apical membrane Na(+)-H(+) exchanger isoform NHE3, we generated monoclonal antibodies (mAbs) against affinity-purified NHE3 protein complexes isolated from solubilized renal microvillus membrane vesicles. Hybridomas were selected based on their ability to immunoprecipitate NHE3. We have characterized in detail one of the mAbs (1D11) that specifically co-precipitated NHE3 but not villin or NaPi-2. Western blot analyses of microvillus membranes and immunoelectron microscopy of kidney sections showed that mAb 1D11 recognizes a 110-kDa protein highly expressed on the apical membrane of proximal tubule cells. Immunoaffinity chromatography was used to isolate the antigen against which mAb 1D11 is directed. N-terminal sequencing of the purified protein identified it as dipeptidyl peptidase IV (DPPIV) (EC ), which was confirmed by assays of DPPIV enzyme activity. We also evaluated the distribution of the NHE3-DPPIV complex in microdomains of rabbit renal brush border. In contrast to the previously described NHE3-megalin complex, which principally resides in a dense membrane population (coated pits) in which NHE3 is inactive, the NHE3-DPPIV complex was predominantly in the microvillar fraction in which NHE3 is active. Serial precipitation experiments confirmed that anti-megalin and anti-DPPIV antibodies co-precipitate different pools of NHE3. Taken together, these studies revealed an unexpected association of the brush border Na(+)-H(+) exchanger NHE3 with dipeptidyl peptidase IV in the proximal tubule. These findings raise the possibility that association with DPPIV may affect NHE3 surface expression and/or activity.

Structural and functional lesions in brush border of human polarized intestinal Caco-2/TC7 cells infected by members of the Afa/Dr diffusely adhering family of Escherichia coli.

Diffusely adhering Escherichia coli (DAEC) strains expressing F1845 fimbrial adhesin or Dr hemagglutinin belonging to the Afa/Dr family of adhesins infect cultured polarized human intestinal cells through recognition of the brush border-associated decay-accelerating factor (DAF; CD55) as a receptor. The wild-type Afa/Dr DAEC strain C1845 has been shown to induce brush border lesions by an adhesin-dependent mechanism triggering apical F-actin rearrangements. In the present study, we undertook to further characterize cell injuries following the interaction of wild-type Afa/Dr DAEC strains C1845 and IH11128 expressing fimbrial F1845 adhesin and Dr hemagglutinin, respectively, with polarized, fully differentiated Caco-2/TC7 cells. In both cases, bacterium-cell interaction was followed by rearrangement of the major brush border-associated cytoskeletal proteins F-actin, villin, and fimbrin, proteins which play a pivotal role in brush border assembly. In contrast, distribution of G-actin, actin-depolymerizing factor, and tubulin was not modified. Using draE mutants, we found that a mutant in which cysteine replaces aspartic acid at position 54 conserved binding capacity but failed to induce F-actin disassembly. Accompanying the cytoskeleton injuries, we found that the distribution of brush border-associated functional proteins sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPPIV), glucose transporter SGLT1, and fructose transporter GLUT5 was dramatically altered. In parallel, SI and DPPIV enzyme activity decreased.

The structure and function of CD26 in the T-cell immune response.

CD26 is a widely distributed 110 kD cell-surface glycoprotein with known dipeptidyl-peptidase IV (DPP-IV) activity in its extracellular domain. This ecto-enzyme is capable of cleaving amino terminal dipeptides from polypeptides with either L-proline or L-alanine in the penultimate position. On human T cells, CD26 expression appears late in thymic differentiation and is preferentially restricted to the CD4+ helper/memory population, and CD26 can deliver a potent co-stimulatory T-cell activation signal. The cDNA sequence of CD26 predicts a type II membrane protein with only 6 amino acids in its cytoplasmic region, suggesting that, in addition to DPP-IV enzyme activity, other signal-inducing molecules may be associated with CD26. Considerable evidence exists that CD26 interacts, presumably in its extracellular domain, with both CD45, a protein tyrosine phosphatase, and adenosine deaminase (ADA), each of which is capable of functioning in a signal transduction pathway. In addition, CD26 is the receptor for ADA, and ADA on the cell surface is involved in an important immunoregulatory mechanism by which released ADA binds to the cell-surface ADA. This multifunctional molecule may be involved in cell migration and the HIV-1-associated loss of CD4+ cells through the process of programmed cell death. Thus, CD26 appears to play a key role in a number of aspects of lymphocyte function.

Dipeptidyl peptidase IV is down-regulated in rat hepatoma cells at the mRNA level.

Dipeptidyl peptidase IV (DPP-IV) is a cell surface ectopeptidase that has been implicated in cell-extracellular matrix interactions, lymphocyte growth and the regulation of biological peptides. Previous studies have shown that immunostaining for DPP-IV and DPP-IV enzyme levels is decreased in hepatoma cells and levels have been correlated with the ability of such cells to adhere in vitro. The aim of this paper was to measure DPP-IV enzyme levels in rat hepatoma cells and to examine whether changes were associated with alterations at the mRNA level. The results indicate a greater than 90% reduction in DPP-IV enzyme levels in two rat hepatoma cell lines, HTC and H35, compared with rat hepatocytes. Enzyme levels of the control enzyme leucine aminopeptidase (LAP) were not decreased. mRNA studies indicated that these changes were associated with similar reductions in rat DPP-IV mRNA. It is concluded that DPP-IV is markedly reduced at the protein, enzyme and mRNA levels in rat hepatoma cells. The significance of these changes is unclear but may lead to decreased extracellular matrix interactions by such cells.