The dipeptidyl peptidase IV (DPIV) enzyme family contains both potential and proven therapeutic targets. Recent reports indicate the presence of DP8 and DP9 in peripheral blood lymphocytes, testis, lung, and brain. For a more comprehensive understanding of DP8 and DP9 tissue and cellular expression, mRNA and enzyme activity were examined. Many organs from C57BL/6 wild-type and DPIV gene-knockout mice were examined; DP8/9 enzyme activity was detected in the immune system, brain, testis, muscle, and epithelia. In situ hybridization localized DP8 and DP9 mRNA to lymphocytes and epithelial cells in liver, gastrointestinal tract, lymph node, spleen, and lung. DP8 and DP9 mRNA was detected in baboon and mouse testis, and DP9 expression was elevated in human testicular cancers. DP8 and DP9 mRNA were ubiquitous in day 17 mouse embryo, with greatest expression in epithelium (skin and gastrointestinal tract) and brain. Thus, DP8 and DP9 are widely expressed enzymes. Their expression in lymphocytes and epithelia indicates potential for roles in the digestive and immune systems. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis.

Dipeptidyl Peptidase (DPP) 4 and related dipeptidyl peptidases are emerging as current and potential therapeutic targets. DPP9 is an intracellular protease that is regulated by redox status and by SUMO1. DPP9 can influence antigen processing, epidermal growth factor (EGF)-mediated signaling and tumor biology. We made the first gene knock-in (gki) mouse with a serine to alanine point mutation at the DPP9 active site (S729A). Weaned heterozygote DPP9wt/S729A pups from 110 intercrosses were indistinguishable from wild-type littermates. No homozygote DPP9S729A/S729A weaned mice were detected. DPP9S729A/S729A homozygote embryos, which were morphologically indistinguishable from their wild-type littermate embryos at embryonic day (ED) 12.5 to ED 17.5, were born live but these neonates died within 8 to 24 hours of birth. All neonates suckled and contained milk spots and were of similar body weight. No gender differences were seen. No histological or DPP9 immunostaining pattern differences were seen between genotypes in embryos and neonates. Mouse embryonic fibroblasts (MEFs) from DPP9S729A/S729A ED13.5 embryos and neonate DPP9S729A/S729A mouse livers collected within 6 hours after birth had levels of DPP9 protein and DPP9-related proteases that were similar to wild-type but had less DPP9/DPP8-derived activity. These data confirmed the absence of DPP9 enzymatic activity due to the presence of the serine to alanine mutation and no compensation from related proteases. These novel findings suggest that DPP9 enzymatic activity is essential for early neonatal survival in mice.

Liver fibrosis is a progressive pathological process involving inflammation and extracellular matrix deposition. Dipeptidyl peptidase 4 (DPP4), also known as CD26, is a cell surface glycoprotein and serine protease. DPP4 binds to fibronectin, can inactivate specific chemokines, incretin hormone and neuropeptides, and influences cell adhesion and migration. Such properties suggest a pro-fibrotic role for this peptidase but this hypothesis needs in vivo examination. Experimental liver injury was induced with carbon tetrachloride (CCl) in DPP4 gene knockout (gko) mice. DPP4 gko had less liver fibrosis and inflammation and fewer B cell clusters than wild type mice in the fibrosis model. DPP4 inhibitor-treated mice also developed less liver fibrosis. DNA microarray and PCR showed that many immunoglobulin (Ig) genes and some metabolism-associated transcripts were differentially expressed in the gko strain compared with wild type. CCl-treated DPP4 gko livers had more IgM and IgG intrahepatic lymphocytes, and fewer CD4, IgD and CD21 intrahepatic lymphocytes. These data suggest that DPP4 is pro-fibrotic in CCl-induced liver fibrosis and that the mechanisms of DPP4 pro-fibrotic action include energy metabolism, B cells, NK cells and CD4 cells.

No abstract

No abstract

No abstract

In recent years, the dipeptidyl peptidase-4 (DPP-4) enzyme family has created intense pharmaceutical interest. DPP-4 inhibitors have proven successful as a therapy for the growing type 2 diabetes epidemic and have potential to treat other diseases. A large number of recently developed DPP-4 inhibitors are in various phases of clinical development, with four gliptin class inhibitors already in clinical use. The unique distribution of Fibroblast Activation Protein (FAP), the closest relative of DPP-4, has led to numerous investigations of it as a target and marker for epithelial cancers. The roles of the newer members, DPP-8 and DPP-9, are yet to be fully characterized, but early evidence suggests possible roles in various aspects of cell biology and disease. There are a number of favorable circumstances that have contributed to the therapeutic approaches of targeting DPP-4 and FAP. Specific targeting of DPP-4 and FAP is made easier by the small size of this enzyme family and several structural differences at their active sites. Additionally, observations over the past decade that the DPP-4 and FAP gene knockout mice are healthy suggests that selective inhibition of each of these proteases would be safe, and this is reflected in the excellent safety profiles of the DPP-4-selective gliptins. These proteins also have interesting extra-enzymatic activities that are expected to be retained in the presence of protease inhibition. This feature also points to a low likelihood of off-target effects. Thus, an overall understanding of DPP-4 and FAP structure–function relationships, distribution, and enzymatic and extra-enzymatic biological roles provides an insight into their therapeutic usefulness as disease targets.