Expression, subcellular localisation, and possible roles of dipeptidyl peptidase 9 (DPP9) in murine macrophages.

Dipeptidyl peptidase 9 (DPP9) is a peptidase of the DPPIV gene family, and its role in immune responses has been reported. In this study, we compared the messenger RNA expression profile of DPP9 to that of the related DPP8 and DPPIV in murine haematopoietic and lymphatic tissues. A similar order of expression levels was observed for all 3 peptidases: peritoneal macrophages < bone marrow < spleen ≤ lymph nodes. Also, we examined the subcellular localisation of DPP9 and its possible role(s) in J774 cell line of macrophage origin. DPP9 was dominantly expressed intracellularly. DPPIV-like enzymatic activity was mostly present in cytoplasm, but also in cell membranes and organelles/vesicles. Decreased expression of DPP9 was observed upon activation of J774 cells by combined treatment with interferon gamma and lipopolysaccharide. Changes induced by DPP9 gene silencing in J774 cells suggest possible role of DPP9 in regulation of proliferation and activation status. The colocalisation of DPP9 with endocytosed DQ-OVA demonstrated in endosomes of J774 cells might suggest the role of DPP9 in peptide processing within endosomal/vesicular compartment.

Dipeptidyl peptidase 9 (DPP9) in human skin cells.

BACKGROUND: Dipeptidyl peptidase 9 (DPP9) is a relatively new member of the DPPIV family of prolyl dipeptidases which is ubiquitously expressed. Its role in regulation of immune responses and proliferation of epithelial carcinoma cells was reported. There is no data on possible role of DPP9 expressed in skin epithelial cells (keratinocytes) and in dermal fibroblasts. MATERIALS AND METHODS: Transcriptional and protein expression of DPP9 and DPPIV was examined in fibroblasts and keratinocytes isolated from normal human skin. Localization of DPP9 and its sub-localization in Golgi were determined by immunocytochemistry staining. DPPIV-like enzyme activity was determined in cell lysates and in isolated cell fractions containing membranes (M), cytosol (C) and content of organelles/endosomes/vesicles (V). Relative contribution of DPPIV and DPP8/9 enzyme activity in these fractions was determined by using selective inhibitors: sitagliptin (selective for DPPIV) and 1G244 (selective for DPP9 and a highly homologous DPP8). Possible roles of DPP8/9 via its enzyme activity were analysed by assessment of survival and proliferative capacity of fibroblasts and HaCaT cells of keratinocyte origin in the presence of the inhibitors. Possible role of DPP9 in cell migration and/or adhesion was analysed in fibroblasts and HaCaT cells after DPP9 gene silencing. RESULTS: Fibroblasts and keratinocytes exerted comparable level of DPP9 both at transcriptional and protein level. Fibroblasts strongly expressed DPPIV, whereas in keratinocytes DPPIV expression was low. DPP9 expression was found in cytosol and in perinuclear area of some fibroblasts, or in scattered pattern of keratinocytes, as well as in nuclei of some cells. Only low level of DPP9 sub-localization within Golgi was observed in fibroblasts and keratinocytes. DPPIV-like enzyme activity was about 5 times higher in lysates of fibroblasts than of HaCaT cells. In fibroblasts DPPIV-like enzyme activity was mainly (65%) found in the fraction containing cell membranes (M) and was predominantly (86.9%) due to DPPIV. In contrast, in HaCaT cells the DPPIV-like enzyme activity was mainly (84.2%) found in cytosol (C) and was predominantly (95.6%) due to DPP8/9. Survival and the proliferative capacity were significantly diminished in the presence of 10µM 1G244, both in fibroblasts and in HaCaT cells, suggesting possible role of DPP8/9 enzyme activity in regulation of survival and proliferation of these cells. DPP9 gene silencing resulted in decreased adhesion of fibroblasts, as well as in decreased migration of fibroblasts and HaCaT cells. Accumulation of DPP9 on the edges of plasma membranes of fibroblasts and keratinocytes adhering to surface supports the idea of possible role of DPP9 in cell adhesion. CONCLUSIONS: This is the first study showing protein expression, sub-localization and possible biological roles of DPP9 expressed in isolated human skin cells. The data may be relevant for development of new drugs against skin diseases by targeting DPP9 expressed in the skin cells.

IFN-γ up-regulates κ opioid receptors (KOR) on murine macrophage cell line J774.

In this study we examined basal and IFN-γ-regulated expression of kappa opioid receptors (KOR) on cells of a murine macrophage cell line, J774. Basal KOR expression was found both at transcriptional and protein levels. KOR protein was predominantly located intracellular (86.4±12.9% positive cells; n=4), and only in minority of J774 cells (9.1±6.4% positive cells; n=4) on plasma membranes, as revealed by Fluorescence-Activated Cell Sorter (FACS) analysis and immunocytochemistry. Proinflammatory cytokine IFN-γ up-regulated KOR expression both at transcriptional (up to 24 times) and protein levels (up to 4.2 times). KOR expressed on J774 cells was functionally active as its ligation with Dynorphin-A(1-17) (100 nM and 1 µM) triggered phosphorylation of ERK1/2. Involvement of KOR in the Dynorphin-A(1-17)-induced triggering of ERK1/2 phosphorylation is suggested since truncated Dynorphin-A(2-17), which does not bind to KOR, was ineffective. Collectively, we have shown for the first time that cells of J774 cell line constitutively express functionally active KOR, which triggers signalization via ERK1/2 phosphorylation and which could be up-regulated by proinflammatory IFN-γ. The data may be relevant for better understanding of the role of KOR and their endogenous ligand Dynorphin-A in regulation of inflammatory and immune responses.

Expression, regulation and functional activities of aminopeptidase N (EC 3.4.11.2; APN; CD13) on murine macrophage J774 cell line.

Aminopeptidase N (APN; CD13) is a ubiquitous membrane-bound enzyme. Expressed on haematopoietic cells APN participates in inflammatory and immune responses by regulating local concentration of chemotactic peptides and by fine-tuning antigen presentation. The data of this study have shown for the first time that cells of murine macrophage line, J774, often used as a model cell line, express CD13 both at transcriptional level and at the level of membrane protein with aminopeptidase N (APN) activity. The level of transcriptional expression of CD13/APN on J774 cells was compared to that found on normal cells participating in immune responses. The highest CD13/APN level was found in peritoneal macrophages, followed by J774 cells and splenocytes, whereas lymph node, thymus and bone-marrow cells expressed low level of CD13/APN mRNA. Further, the CD13 (mRNA, protein and APN) on J774 cells could be up-regulated by pro-inflammatory IFN-γ which is in agreement with the known role of CD13/APN in inflammatory responses. Co-regulation of CD13 with MHC-II and CD86 is in line with the reported role of APN expressed on human cells in antigen presentation. CD13 on J774 cells co-localize with mannose receptors (MR), and co-internalize upon MR ligation by ovalbumin, suggesting a new function of CD13 in MR-mediated phagocytosis. That function of CD13 is independent of APN enzyme activity. Anti-inflammatory drug dexamethasone diminished the IFN-γ-induced increase of CD13. The observed down-regulation of CD13 on J774 cells by dexamethasone might be relevant as a possible mechanism involved in action of anti-inflammatory drugs on normal macrophages.

Regulation of aminopeptidase N (EC 3.4.11.2; APN; CD13) on the HL-60 cell line by TGF-beta(1).

Membrane-bound peptidases interfere with cellular growth, differentiation, activation and death by fine-tuning local concentrations of various signaling peptides such as the growth factors, hormones, chemokines and cytokines. We examined the effects of anti-inflammatory cytokine transforming growth factor-beta(1) (TGF-beta(1)) on the expression and activity of aminopeptidase N (APN), an ectoenzyme processing several signaling peptides. Myelo-monocytic HL-60 cell line having high basal APN activity corresponding to the membrane CD13 marker served as a model. Regulation of CD13/APN was assayed at the levels of mRNA and at the membrane marker CD13. Functional properties of CD13/APN were examined by measuring the enzyme activity, and the signal transduction ability, followed as Ca(++) mobilization triggered by APN-blocking WM-15 antibody. TGF-beta(1) at physiological concentrations (0.16 to 2.5 ng/mL) increased expression of CD13 both at mRNA and membrane protein level in a time- and concentration-dependent manner. Transcriptional activation of CD13 by TGF-beta(1) is suggested as actinomycin-D, an inhibitor of RNA synthesis, abrogated the TGF-beta(1)-induced up-regulation of CD13. Increased membrane CD13 expression was associated with an increase of its enzyme (APN) activity and with a decrease of its signal transduction ability. Anti-inflammatory cytokine TGF-beta(1) counteracted the effects of pro-inflammatory cytokine IFN-gamma on membrane CD13 expression in a time- and concentration-dependent fashion, suggesting a cytokine-regulated role of CD13/APN in inflammation. This is the first report on regulation of CD13/APN expression by TGF-beta(1) on immature cells of myelo-monocytic origin. As obtained with physiological concentrations of TGF-beta(1) these findings may be relevant for cytokine-regulated CD13/APN expression on mature myeloid cells in the course of inflammation.

Receptor-mediated down-regulation of neutral endopeptidase (NEP; EC 3.4.24.11; CD10) on immature B lymphocytes by dexamethasone.

Neutral endopeptidase is a membrane bound enzyme with various functions depending on cell type or tissue origin. Normal development and differentiation of immature B lymphocytes depends on expression of CD10/NEP on B cell progenitors and bone marrow stromal cells. Synthetic glucocorticoid dexamethasone (dex), an immunosuppressive and anti-inflammatory drug, was shown to be a potent modulator of CD10/NEP expressed on cells of non-hematopoietic origin. We investigated the effect of dex on expression of differentiation marker CD10/NEP on immature B cells. The drug was applied in concentrations corresponding to the physiological range. CD10/NEP was measured at three levels of expression: mRNA (by means of duplex PCR), membrane protein marker (FACS analysis) and enzyme activity (hydrolysis of a selective chromogenic substrate). Dex down-regulated CD10/NEP expression on immature B cell line NALM-6 in a concentration- and time-dependent fashion. The effect was detected at all three levels. Dex-induced CD10/NEP down-regulation was mediated via glucocorticoid receptors (GR), as it was fully abrogated by a GR antagonist, RU 38486. That occurred at all three levels. The mechanism of dex-induced CD10/NEP down-regulation is not likely to include selection of cells that are CD10low since the effect was partly reversible after the removal of dex. However, dex-induced CD10/NEP down-regulation did include decreased transcription of the CD10 mRNA. Transcriptional inhibitor actinomycin D completely abolished dex-induced CD10/NEP down-regulation. Since differentiation of normal B lymphocytes is associated with down-regulation of CD10/NEP, the data presented suggest that low, physiologically relevant concentrations of glucocorticoids (such as observed in acute stress) may play a regulatory role in normal development and maturation of B lymphocytes.

Regulation of aminopeptidase N (EC 3.4.11.2; APN; CD13) by interferon-gamma on the HL-60 cell line.

Membrane-bound peptidases play important roles in the regulation of local concentrations of various signalling peptides such as the growth factors, hormones, chemokines and cytokines. That is accomplished by means of their enzyme activity. Recently, membrane-bound peptidases have also been shown to act as receptors, receiving signals from as yet undefined ligands and transducing them into the cell interior. By using either or both of these mechanisms, peptidases interact with fundamental cellular functions: growth, differentiation, activation and death. This study addressed the effects of a T-cell derived cytokine, interferon-gamma (IFN-gamma) on the activity of aminopeptidase N (APN), an ectoenzyme processing several signal peptides. Cells of a myelo-monocytic cell line HL-60 were used as a model system, and APN was assayed at the levels of mRNA, its membrane marker CD13, and the enzyme activity. Regulation of CD13/APN by IFN-gamma was found at all three levels. The direction of regulation was time-dependent: an initial down-regulation seen 24 and 48 hrs after the onset of treatment with IFN-gamma was replaced by an up-regulation after 72 and/or 96 hrs. Up-regulation of CD13/APN observed after 96 hrs was preceded by an up-regulation of APN mRNA reaching its maximum after 72 hrs. The IFN-gamma-induced regulation of APN was due to membrane aminopeptidase N, since it could be completely abrogated by an APN blocking antibody WM-15. The delayed up-regulation of CD13/APN (observed after 72 and/or 96 hrs), required de novo protein synthesis as it could be abrogated by cycloheximide, an inhibitor of protein synthesis. Possible role of endogenous (IFN-gamma-induced) TGF-beta in mediating CD13/APN up-regulation could be excluded, since no TGF-beta was found in supernatants of IFN-gamma treated HL-60 cells. Thus, our data show regulation of CD13/APN on cells of myelo-monocytic origin by a T-cell derived cytokine, IFN-gamma. A similar mechanism might play a role in inflammation.

Expression of CD13/aminopeptidase N and CD10/neutral endopeptidase on cultured human keratinocytes.

Keratinocytes actively participate in immune response and inflammation by secreting cytokines and chemokines. Membrane-bound peptidases serve as negative loop in controlling concentration of peptide signalling molecules. Recently, they have also been proposed as additional mechanism of cell-to-cell interaction and as signalling molecules. In this study, we examined expression of two membrane-bound peptidases: aminopeptidase N (APN; EC 3.4.11.2; CD13) and neutral endopeptidase (NEP; EC 3.4.24.11; CD10) on nonstimulated cultured human keratinocytes obtained from healthy skin. Membrane expression of CD13 and CD10 was analysed by FACS and fluorescent microscope. Functional properties of CD13 and CD10 were examined by testing their enzymatic activity towards selective substrates. The data were compared to those obtained on cultured nonstimulated human skin fibroblasts expressing both CD13/APN and CD10/NEP. Approximately one-third (i.e. 31.7+/-2.8%; n=3) of cultured keratinocyte express CD13 as compared to fibroblasts which are 100% CD13(+) (n=3). Density of CD13 on keratinocytes is several times lower than on fibroblasts. Membrane CD13 expression on keratinocytes was associated with significant enzyme activity, which on the basis of substrate (L-Ala-betaNA) and inhibitor (bestatin, actinonin) selectivity could be ascribed to aminopeptidase N. Kinetic parameter V(max) revealed lower APN activity expressed on keratinocytes than on fibroblasts (V(max)=1.49+/-0.08 microM/60 min/5 x 10(4) cells for keratinocytes, n=3 versus V(max)=4.09+/-0.76 microM/60 min/5 x 10(4) cells for fibroblasts, n=3). Likewise, K(m) value of APN on keratinocytes was lower as compared to fibroblasts (K(m)=0.307+/-0.090 mM for keratinocytes, n=3 versus K(m)=0.766+/-0.065 mM for fibroblasts, n=3). CD13 demonstrated on cultured keratinocytes, is at least partly due to its constitutive expression since it was also found on freshly prepared epidermal skin cells. Inhibitors of APN, actinonin, bestatin and substance-P, as well as the APN blocking antibody WM-15, decreased keratinocytes growth. In contrast to membrane CD13 associated with APN enzyme activity, neither membrane CD10, nor its enzyme (NEP) activity could be found on the same keratinocyte samples. In conclusion, functional CD13, associated with APN activity, was found on about one third of cultured, non-stimulated keratinocytes, whereas no CD10/NEP was found on the same keratinocyte samples. Role of APN in regulation of keratinocyte growth is suggested, as its inhibition resulted in decreased keratinocyte growth.