Transmembrane prostatic acid phosphatase (TMPAP) delays cells in G1 phase of the cell cycle.

BACKGROUND: Prostate adenocarcinoma is the most common form of prostate cancer. We have previously shown in a murine model that prostatic acid phosphatase (PAP) deficiency leads to increased cell proliferation and development of prostate adenocarcinoma. The association between PAP and prostate cancer has been reported. Indeed, high PAP enzymatic activity is detected in the serum of patients with metastatic disease while its expression is reduced in prostate cancer tissue. However, the molecular mechanisms behind the onset of the disease remains poorly understood. We previously identified a novel transmembrane prostatic acid phosphatase (TMPAP) isoform, which interacts with snapin. TMPAP is expressed on the plasma membrane, as well as endosomal/lysosomal and exosomal membrane vesicles by means of a tyrosine-based lysosomal targeting motif (Yxxϕ). METHODS: We used stable overexpression of the secreted isoform (SPAP) and TMPAP in LNCaP cells, live cell imaging, microarray and qRT-PCR analyses, and fluid phase uptake of HRP and transferrin. RESULTS: Our results indicate that the stable overexpression of TMPAP, but not SPAP in LNCaP cells reduces cell growth while increasing endo/exocytosis and cell size. Specifically, cells overexpressing TMPAP accumulate in the G1 phase of the cell cycle, and show altered gene expression profile. CONCLUSIONS: Our data suggests that TMPAP may function as a non-canonical tumor suppressor by delaying cell growth in G1 phase of the cell cycle.

Prostatic acid phosphatase is the main acid phosphatase with 5'-ectonucleotidase activity in the male mouse saliva and regulates salivation.

We have previously shown that in addition to the well-known secreted isoform of prostatic acid phosphatase (sPAP), a transmembrane isoform exists (TMPAP) that interacts with snapin (a SNARE-associated protein) and regulates the endo-/exocytic pathways. We have also shown that PAP has 5'-ectonucleotidase and thiamine monophosphatase activity and elicits antinociceptive effects in mouse models of chronic inflammatory and neuropathic pain. Therefore, to determine the physiological role of PAP in a typical exocrine organ, we studied the submandibular salivary gland (SMG) of PAP(-/-) and wild-type C57BL/6J mice by microarray analyses, microRNA sequencing, activity tests, immunohistochemistry, and biochemical and physiological analyses of saliva. We show that PAP is the main acid phosphatase in the wild-type male mouse saliva, accounting for 50% of the total acid phosphatase activity, and that it is expressed only in the granular convoluted tubules of the SMGs, where it is the only 5'-ectonucleotidase. The lack of PAP in male PAP(-/-) mice was associated with a significant increase in the salivation volume under secretagogue stimulation, overexpression of genes related to cell proliferation (Mki67, Aurkb, Birc5) and immune response (Irf7, Cxcl9, Ccl3, Fpr2), and upregulation of miR-146a in SMGs. An increased and sustained acinar cell proliferation was detected without signs of glandular hyperplasia. Our results indicate that in PAP(-/-) mice, SMG homeostasis is maintained by an innate immune response. Additionally, we suggest that in male mice, PAP via its 5'-ectonucleotidase activity and production of adenosine can elicit analgesic effects when animals lick their wounds.

Prostatic acid phosphatase is not a prostate specific target.

Prostatic acid phosphatase (PAP) is currently evaluated as a target for vaccine immunotherapy of prostate cancer. This is based on the previous knowledge about secretory PAP and its high prostatic expression. We describe a novel PAP spliced variant mRNA encoding a type I transmembrane (TM) protein with the extracellular NH(2)-terminal phosphatase activity and the COOH-terminal lysosomal targeting signal (YxxPhi). TM-PAP is widely expressed in nonprostatic tissues like brain, kidney, liver, lung, muscle, placenta, salivary gland, spleen, thyroid, and thymus. TM-PAP is also expressed in fibroblast, Schwann, and LNCaP cells, but not in PC-3 cells. In well-differentiated human prostate cancer tissue specimens, the expression of secretory PAP, but not TM-PAP, is significantly decreased. TM-PAP is localized in the plasma membrane-endosomal-lysosomal pathway and is colocalized with the lipid raft marker flotillin-1. No cytosolic PAP is detected. We conclude that the wide expression of TM-PAP in, for instance, neuronal and muscle tissues must be taken into account in the design of PAP-based immunotherapy approaches.

Mice deficient in transmembrane prostatic acid phosphatase display increased GABAergic transmission and neurological alterations.

Prostatic acid phosphatase (PAP), the first diagnostic marker and present therapeutic target for prostate cancer, modulates nociception at the dorsal root ganglia (DRG), but its function in the central nervous system has remained unknown. We studied expression and function of TMPAP (the transmembrane isoform of PAP) in the brain by utilizing mice deficient in TMPAP (PAP-/- mice). Here we report that TMPAP is expressed in a subpopulation of cerebral GABAergic neurons, and mice deficient in TMPAP show multiple behavioral and neurochemical features linked to hyperdopaminergic dysregulation and altered GABAergic transmission. In addition to increased anxiety, disturbed prepulse inhibition, increased synthesis of striatal dopamine, and augmented response to amphetamine, PAP-deficient mice have enlarged lateral ventricles, reduced diazepam-induced loss of righting reflex, and increased GABAergic tone in the hippocampus. TMPAP in the mouse brain is localized presynaptically, and colocalized with SNARE-associated protein snapin, a protein involved in synaptic vesicle docking and fusion, and PAP-deficient mice display altered subcellular distribution of snapin. We have previously shown TMPAP to reside in prostatic exosomes and we propose that TMPAP is involved in the control of GABAergic tone in the brain also through exocytosis, and that PAP deficiency produces a distinct neurological phenotype.

Purification of prostatic acid phosphatase (PAP) for structural and functional studies.

High-scale purification methods are required for several protein studies such as crystallography, mass spectrometry, circular dichroism, and function. Here we describe a purification method for PAP based on anion exchange, L-(+)-tartrate affinity, and gel filtration chromatographies. Acid phosphatase activity and protein concentration were measured for each purification step, and to collect the fractions with the highest acid phosphatase activity the p-nitrophenyl phosphate method was used. The purified protein obtained by the procedure described here was used for the determination of the first reported three-dimensional structure of prostatic acid phosphatase.

Transmembrane prostatic acid phosphatase (TMPAP) interacts with snapin and deficient mice develop prostate adenocarcinoma.

The molecular mechanisms underlying prostate carcinogenesis are poorly understood. Prostatic acid phosphatase (PAP), a prostatic epithelial secretion marker, has been linked to prostate cancer since the 1930's. However, the contribution of PAP to the disease remains controversial. We have previously cloned and described two isoforms of this protein, a secretory (sPAP) and a transmembrane type-I (TMPAP). The goal in this work was to understand the physiological function of TMPAP in the prostate. We conducted histological, ultra-structural and genome-wide analyses of the prostate of our PAP-deficient mouse model (PAP(-/-)) with C57BL/6J background. The PAP(-/-) mouse prostate showed the development of slow-growing non-metastatic prostate adenocarcinoma. In order to find out the mechanism behind, we identified PAP-interacting proteins byyeast two-hybrid assays and a clear result was obtained for the interaction of PAP with snapin, a SNARE-associated protein which binds Snap25 facilitating the vesicular membrane fusion process. We confirmed this interaction by co-localization studies in TMPAP-transfected LNCaP cells (TMPAP/LNCaP cells) and in vivo FRET analyses in transient transfected LNCaP cells. The differential gene expression analyses revealed the dysregulation of the same genes known to be related to synaptic vesicular traffic. Both TMPAP and snapin were detected in isolated exosomes. Our results suggest that TMPAP is involved in endo-/exocytosis and disturbed vesicular traffic is a hallmark of prostate adenocarcinoma.