Rolf Mentlein

Dipeptidyl-peptidase IV (CD26)-role in the inactivation of regulatory peptides

Dipeptidyl-peptidase IV (DPP IV/CD26) has a dual function as a regulatory protease and as a binding protein. Its role in the inactivation of bioactive peptides was recognized 20 years ago due to its unique ability to liberate Xaa-Pro or Xaa-Ala dipeptides from the N-terminus of regulatory peptides, but further examples are now emerging from in vitro and vivo experiments. Despite the minimal N-terminal truncation by DPP IV, many mammalian regulatory peptides are inactivated--either totally or only differentially--for certain receptor subtypes. Important DPP IV substrates include neuropeptides like neuropeptide Y or endomorphin, circulating peptide hormones like peptide YY, growth hormone-releasing hormone, glucagon-like peptides(GLP)-1 and -2, gastric inhibitory polypeptide as well as paracrine chemokines like RANTES (regulated on activation normal T cell expressed and secreted), stromal cell-derived factor, eotaxin and macrophage-derived chemokine. Based on these findings the potential clinical uses of selective DPP IV inhibitors or DPP IV-resistant analogues, especially for the insulinotropic hormone GLP-1, have been tested to enhance insulin secretion and to improve glucose tolerance in diabetic animals. Thus, DPP IV appears to be a major physiological regulator for some regulatory peptides, neuropeptides, circulating hormones and chemokines.

Proteolytic processing of neuropeptide Y and peptide YY by dipeptidyl peptidase IV

Neuropeptide Y, peptide YY and pancreatic polypeptide share an evolutionary conserved proline-rich N-terminal sequence, a structure generally known to be inert to the attack of common proteinases, but a potential target for specialized proline-specific aminopeptidases. Purified human dipeptidyl peptidase IV (also termed CD 26) liberated N-terminal Tyr-Pro from both, neuropeptide Y and peptide YY, with very high specific activities and Km values in the micromolar range, but almost no Ala-Pro from pancreatic polypeptide. Other proline-specific aminopeptidases exhibited low (aminopeptidase P, liberation of N-terminal Tyr) or totally no activity (dipeptidyl peptidase II), as was also observed with less-specific aminopeptidases (aminopeptidase M, leucine aminopeptidase). When human serum was incubated with neuropeptide Y or peptide YY at micro- and nanomolar concentrations, Tyr-Pro was detected as a metabolite of both peptides. Formation of Tyr-Pro in serum was blocked in the presence of Lys-pyrrolidine and diprotin A (Ile-Pro-Ile), specific, competitive inhibitors of dipeptidyl peptidase IV. Incubation of neuropeptide Y or peptide YY with immunocytochemically defined, cultivated endothelial cells from human umbilical cord also yielded Tyr-Pro. Dipeptidyl peptidase IV could be immunostained on most endothelial cells by a specific antibody. We suggest that dipeptidyl peptidase IV might be involved in the degradation of neuropeptide Y and peptide YY to N-terminal truncated neuropeptide Y(3-36) and peptide YY(3-36). Since specific binding to Y1, but not to Y2 subtype of neuropeptide Y/peptide YY receptors requires intact N- as well as C-termini of neuropeptide Y and peptide YY, removal of their amino-terminal dipeptides by dipeptidyl peptidase IV inactivates them for binding to one receptor subtype.

The transmembrane CXC-chemokine ligand 16 is induced by IFN-gamma and TNF-alpha and shed by the activity of the disintegrin-like metalloproteinase ADAM10.

The novel CXC-chemokine ligand 16 (CXCL16) functions as transmembrane adhesion molecule on the surface of APCs and as a soluble chemoattractant for activated T cells. In this study, we elucidate the mechanism responsible for the conversion of the transmembrane molecule into a soluble chemokine and provide evidence for the expression and shedding of CXCL16 by fibroblasts and vascular cells. By transfection of human and murine CXCL16 in different cell lines, we show that soluble CXCL16 is constitutively generated by proteolytic cleavage of transmembrane CXCL16 resulting in reduced surface expression of the transmembrane molecule. Inhibition experiments with selective hydroxamate inhibitors against the disintegrin-like metalloproteinases a disintegrin and metalloproteinase domain (ADAM)10 and ADAM17 suggest that ADAM10, but not ADAM17, is involved in constitutive CXCL16 cleavage. In addition, the constitutive cleavage of transfected human CXCL16 was markedly reduced in embryonic fibroblasts generated from ADAM10-deficient mice. By induction of murine CXCL16 in ADAM10-deficient fibroblasts with IFN-γ and TNF-α, we show that endogenous ADAM10 is indeed involved in the release of endogenous CXCL16. Finally, the shedding of endogenous CXCL16 could be reconstituted by retransfection of ADAM10-deficient cells with ADAM10. Analyzing the expression and release of CXCXL16 by cultured vascular cells, we found that IFN-γ and TNF-α synergize to induce CXCL16 mRNA. The constitutive shedding of CXCL16 from the endothelial cell surface is blocked by inhibitors of ADAM10 and is independent of additional inhibition of ADAM17. Hence, during inflammation in the vasculature, ADAM10 may act as a CXCL16 sheddase and thereby finely control the expression and function of CXCL16 in the inflamed tissue.

Proline residues in the maturation and degradation of peptide hormones and neuropeptides

The proteases involved in the maturation of regulatory peptides like those of broader specificity normally fail to cleave peptide bonds linked to the cyclic amino acid proline. This generates several mature peptides with N‐terminal X‐Prosequences. However, in certain non‐mammalian tissues repetitive pre‐sequences of this type are removed by specialized dipeptidyl (amino)peptidases during maturation. In mammals, proline‐specific proteases are not involved in the biosynthesis of regulatory peptides, but due to their unique specificity they could play an important role in the degradation of them. Evidence exists that dipeptidyl (amino)peptidase IV at the cell surface of endothelial cells sequesters circulating peptide hormones which are then susceptible to broader aminopeptidase attack. The cleavage of several neuropeptides by prolyl endopeptidase has been demonstrated in vitro, but its role in the brain is questionable since the precise localization of the protease is not clarified.

The role of vasculature and angiogenesis for the pathogenesis of degenerative tendons disease.

More than 100 years ago Wilhelm Roux (1895) introduced the term “functional adaptation to anatomy and physiology”. Compared with other organ systems the functional adaptation processes are best identifiable in the locomotor system, like for example in the two types of tendons: traction and gliding tendons. Traction tendons are tendons where the direction of pull is in line with the direction of the muscle (e.g. Achilles tendon). Gliding tendons (e.g. tibialis posterior tendon) change direction by turning around a bony or fibrous hypomochlion. In this region the tendon is subjected to intermittent compressive and shear forces and the extracellular matrix consists of avascular fibrocartilage. Avascularity is considered to be a key factor for the etiology of degenerative tendon disease. The repair capability after repetitive microtrauma is strongly compromised in avascular tissue of gliding tendons. Reduced vascularity is not a specific feature of gliding tendons; several studies have shown that the number and size of blood vessels are largely shortened in the waist of the Achilles tendon. However, histological biopsies from degenerated Achilles tendons and Doppler flow examinations revealed a high blood vessel density in patients with degenerative tendon disease.

The splice variants VEGF121 and VEGF189 of the angiogenic peptide vascular endothelial growth factor are expressed in osteoarthritic cartilage

OBJECTIVE Vascular endothelial growth factor (VEGF) has recently been shown to play an important role during endochondral bone formation in hypertrophic cartilage remodeling, ossification, and angiogenesis, but it is not expressed in normal adult cartilage. Since genes expressed during development often reappear in the disease state, we investigated whether VEGF and its receptors (VEGFRs) are expressed in osteoarthritic (OA) cartilage. METHODS VEGF production in OA cartilage from the tibial plateau was measured by enzyme-linked immunosorbent assay. Deposition of VEGF and VEGFR was determined by immunohistochemistry. Expression of messenger RNA for the different VEGF splice forms and for VEGFR was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS Increased VEGF concentrations were measured in OA cartilage from the tibial plateau, while VEGF was almost undetectable in normal cartilage but could be immunostained within the intracellular and pericellular matrices of OA chondrocytes. In analyses of cartilage samples from all 10 OA patients evaluated, VEGF121 and VEGF189 were identified as the only VEGF splice forms expressed. RT-PCR and immunohistochemistry for VEGF in normal hyaline cartilage yielded negative findings. In addition to VEGF, VEGFR-2 (kinase domain region/fetal liver kinase 1), but not VEGFR-1 (fms-like tyrosine kinase 1), could be detected by RT-PCR in OA cartilage and immunostained on OA chondrocytes. CONCLUSION Apart from its production in hypertrophic chondrocytes, VEGF is also produced in chondrocytes of OA cartilage. While the splice variant VEGF189 binds to extracellular matrix proteoglycans, VEGF121 is diffused freely. Both proteins should contribute to the inflammatory process by autocrine/paracrine stimulation of chondrocytes, chemotaxis of macrophages, and promotion of angiogenesis.

Stromal cell-derived factor 1 is secreted by meningeal cells and acts as chemotactic factor on neuronal stem cells of the cerebellar external granular layer.

The cerebellar external granular layer (EGL) is an unusually long-lasting neural proliferative zone positioned immediately beneath the pial surface. Its position and stability critically depend on meningeal cells, as their selective destruction leads to its rapid dispersal, creating massive cortical ectopia. Similar ectopias have recently been described as a side effect of deficiency for stromal cell-derived factor 1 (SDF-1), a chemoattractant for haematopoietic precursor cell migration. Here we show that SDF-1 is present in meningeal cells in vivo and in vitro, where it is secreted in functionally relevant concentrations into the medium. Correspondingly, the SDF-1 receptor (termed CXCR4) can be demonstrated on stem cells of the external granular layer, but is absent on postmitotic cells commencing their final inward migration. We show that SDF-1 is concentrated by heparan sulphate proteoglycans highly expressed in the EGL in a laminar fashion, which thus might act to locally restrict SDF-1 action to the EGL in a kind of step gradient. In vitro, SDF-1 chemotactically attracts neuronal cells isolated from the external, but not from the internal granular layer, in a Boyden chamber assay in concentrations found in meningeal cell-conditioned medium. Selective removal of SDF-1 from conditioned media by immunoprecipitation abolishes their chemoattractive action, which can be reconstituted again by the addition of recombinant SDF-1. Meningeal cells are thus an important source for the expression of SDF-1 during brain development, which--comparable to its role in haematopoiesis--appears to be a key factor attracting precursor cells to their proliferative compartment.

Expression of stem cell markers in human astrocytomas of different WHO grades

According to new hypotheses astrocytomas/gliomas either arise from or attract neural stem cells. Biological markers, particularly antigenic markers, have played a significant role for the characterization of these tumour stem cells (TSCc). Because these studies have been performed with single experimental samples mostly from gliomas, we investigated the expression of the stem cell markers CD133/Prominin, Nestin, Sox-2, Musashi-1, CXCR4, Flt-4/VEGFR-3 and CD105/Endoglin in 72 astrocytomas of different WHO-grades and compared it to normal adult human brain. Expression of their mRNA was quantified by quantitative RT-PCR, of their protein by counting immunopositive cells. In contrast to normal brain, tumour samples showed a high variability for the expression of all markers. However, their mean expression was significantly increased in astrocytomas, but this depended on the WHO grade only for CD133, Nestin, Sox-2 and Musashi-1. Confocal microscopy revealed that these markers mostly could be co-stained with glial fibrillary acidic protein, a marker for astoglial cells, but less frequently with the proliferation marker Ki-67/MIB-1. These markers sometimes, but not necessarily could be co-stained with each other in complex patterns. Our results show that most astrocytomas contain considerable portions of cells expressing stem cell markers. It appears that some of these cells originate from tumour genesis (supporting the stem cell hypothesis) while others are attracted by the tumours. Further functional markers are required to differentiate these TSC-types.

Simultaneous purification and comparative characterization of six serine hydrolases from rat liver microsomes

Abstract Rat liver microsomes contain many serine hydrolases, which can be demonstrated in electropherograms with carboxylesterase stain and with an active-site-directed radioactive organophosphate. Five of the most prominent of these enzymes plus dipeptidyl aminopeptidase IV, a microsomal serine hydrolase without activity against simple esters, have been highly purified with a simultaneous procedure after solubilization with saponin. The five carboxylesterases belong to at least three groups of chemically different proteins. Terminal amino acids, amino acid composition, and substrate specificity are different, while the subunit molecular weight of all esterases is very similar (about 60,000). All purified carboxylesterases have monooleylglycerol-cleaving capacity. The subunit weight (84,000) and the N-terminal amino acid (serine) of the peptidase differ from those of all isolated carboxylesterases. The data are correlated to other reports on individual serine hydrolases from rat liver.

Vascular endothelial growth factor (VEGF) induces matrix metalloproteinase expression in immortalized chondrocytes

VEGF (vascular endothelial growth factor), an important angiogenesis factor, appears also to be involved in inflammatory processes. Recent studies have shown that VEGF and its receptors (VEGFR) are expressed on osteoarthritic, but not on normal adult, chondrocytes. To elucidate possible functions of VEGF in osteoarthritic cartilage, the effects of VEGF were studied on immortalized human chondrocytes. Activated matrix metalloproteinase (MMP)‐1, MMP‐3, MMP‐13, tissue inhibitor of metalloproteinases (TIMP)‐1, TIMP‐2, interleukin (IL)‐1β, IL‐6, and tumour necrosis factor‐α (TNF‐α) were measured in culture supernatants by enzyme‐linked immunosorbent assays, nitric oxide with the Griess reagent, and cell proliferation by [3H]thymidine incorporation. VEGFR‐2 mRNA was quantified by real‐time reverse transcription‐polymerase chain reaction and the protein was identified by immuno‐gold electron microscopy. Intracellular signal transduction effects were determined by western blots and electrophoretic mobility shift assays. The chondrocyte cell lines C28/I2, C20/A4, and T/C28a2/a4 expressed functionally active VEGFR‐2. VEGF stimulation induced receptor phosphorylation, activation of the mitogen‐activated protein kinases ERK 1/2, and long‐lasting activation of the transcription factor AP‐1 (activator protein‐1). VEGF increased secreted MMP‐1, MMP‐3, and especially MMP‐13, which could be effectively reduced by an inhibitor of VEGFR‐2 kinase activity. Interestingly, VEGF diminished the expression of TIMP‐1 and especially TIMP‐2. Under hypoxic conditions, as occur in cartilage, the reduction in TIMP levels was even greater. Furthermore, VEGF induced IL‐1β, IL‐6, TNF‐α, and nitric oxide expression to a small extent and stimulated the proliferation of immortalized chondrocytes. These findings indicate that VEGF is an autocrine stimulator of immortalized chondrocytes that mediates mainly destructive processes in osteoarthritis. Copyright