Transmembrane Prostatic Acid Phosphatase (TMPAP) Interacts with Snapin and Deficient Mice Develop Prostate Adenocarcinoma

Peer reviewe

Mice Deficient in Transmembrane Prostatic Acid Phosphatase Display Increased GABAergic Transmission and Neurological Alterations

Peer reviewe

Prostatic acid phosphatase (PAP) 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) which 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 anti-nociceptive effects in mouse models of chronic inflammatory and neuropathic pain. Therefore, in order 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 it is only expressed in the granular convoluted tubules of the SMGs, where it is the only 5'-ectonucleotidase. The lack of PAP in the 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 SMG. 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

TMPAP co-localize and interact with snapin in the cell lamellipodia.

<p>A, co-localization (yellow) of TMPAP (green) with snapin (red) was observed in the vesicles and lamellipodia of the TMPAP/LNCaP cells. Arrows mark the co-localization points in the upper panel (scale bar: 20 µm). Lower panel (scale bar: 3 µm) showing the lamellipodia region, amplification of the area marked with a box in the upper panel (left). B, intensification of donor (TMPAP-GFP) fluorescence in LNCaP cells was observed after acceptor (snapin-DsRed) photobleaching which confirms FRET between two molecules (Scale bar: 2 µm).</p

DLP lobeexhibits the primary changes in the PAP^−/− mouse prostate.

<p>The panels show an overview of the 12-old mice prostate dissected lobes. The DLP, AP and VP lobes were dissected from WT and PAP^−/− mouse. The monolayer epithelium (white arrows) is seen in all the lobes of the WT mouse, whereas in the PAP^−/− mouse an increased amount of cells is present in the lumen of the DLP lobe (black arrows). The AP and VP of PAP^−/− mouse show no significant changes. Scale bars: 100 µm.</p

The prostate adenocarcinoma in PAP^−/− mice is also detected by immunohistochemistry and electronmicroscopy.

<p>A, smooth muscle actin (SMA) immunohistochemistry in 12 month-old mice. Monolayer epithelium (mL) and open lumen in PAP^+/+ DLP. White arrows show the broken fibromuscular sheath (SM, smooth muscle) and bulging of epithelial cells to the stroma. Prostate adenocarcinoma (black arrows) is present in AP and DLP, showing a multilayer epithelium (ML) and inflammatory cells (black arrowhead) spreading in neighboring areas. Scale bars: 100 µm. (<i>n</i> =  4, per group). B, ultrastructural changes in 3 month-old and 12 month-old PAP^−/− mouse DLPs. Monolayer epithelium, regular basement membrane (BM) and apical secretion are clearly seen in PAP^+/+ mouse DLPs. 3 month-old PAP^−/− DLPs show irregular BM and numerous apical vacuoles (red arrow head), as well the presence of basal lysosomes (Ly). In 12 month-old PAP−/− mouse DLPs, the epithelium has transformed to a multilayer epithelium containing hyperchromatic nuclei with multiple nucleoli. Pseudolumens (pL) have formed as a result of the growing and fusion of the epithelium. Invaginations of BM (red arrows) into the epithelium and numerous vesicles in the basal side of the cells (blue arrow heads) were additional signs of the transformation in the cells. Scale bars: 2000 nm (<i>n</i> = 4, per group).</p

Significant ontological terms obtained with GoMiner software from two-color microarrays experiments.

<p><b>GO ID</b>: gene ontology ID accession number. <b><i>P</i></b><b>-value</b>: <i>P</i>-value for the number of changed genes in the input list, significant <i>P</i>-value <0.05. <b>Term</b>: associated ontological term. <b>Rows in bold</b>: relevant ontological groups for vesicular transport. Mice in microarray experiment per group, <i>n = </i>3.</p

Proliferation of DLP cells is increased in PAP^−/− mice compared to WT mice.

<p>A, bar plot showing the ratio (as percentage) between proliferative cell counts and total amount of cells. (**, P value <0.01; ***, P value <0.001). Error bars indicate S.E.M. values. B, bar plot showing the ratio (as percentage) between apoptotic cell counts and total amount of cells. Error bars indicate S.E.M. values.</p

TMPAP is involved in endo-/exocytosis (proposed mechanism).

<p>TMPAP synthesized in the endoplasmic reticulum is transported in vesicles to the plasma membrane through the trans-Golgi network (TGN). After the vesicle docking and fusion events leading to release of vesicle content, TMPAP inserted in plasma membrane exerts its phosphatase function over AMP. The resulting product adenosine (Ado) activates the adenosine receptors, which are GPCRs, A1 or A3with G_αi (inhibitory G-protein β-subunit) specificity leading to the inhibition of adenylate cyclase (AC) activity, and A2 adenosine receptors with G_αs (stimulatory G-protein α-subunit) producing the stimulation of AC activity. Activated AC produces cAMP, which activates PKA responsible for the phosphorylation of snapin. The turnover is completed by clathrin-mediated endocytosis of SNARE components and TMPAP for recycling and degradation in lysosomes vía the endosomal-lysosomal pathway. From early endosomes, the cargo can be sorted to late endosomes or to MVE, which can follow the route leading to exosome release. Additional dephosphorylation events by TMPAP can occur while trafficking between different compartments. From late endosomes, TMPAP can go to lysosomes or back to TGN via the retrograde pathway. ATP: adenosine triphosphate, ADP: adenosine diphosphate, AMP: adenosine monophosphate, Ado: adenosine, TGN: trans-Golgi network, P: phosphate group, AP-2: adaptor protein complex 2, ADORA: adenosine receptor A (types A1, A2 and A3), AC: adenylate cyclase, G_αs, G_αi, G_β, G_γ: G-protein subunits, VDCC: Voltage-gated calcium channel. Synaptobrevin, syntaxin and SNAP25 are SNARE proteins. PI (4,5) P₂: phosphatidylinositol 4,5-bisphosphate.</p

The colocalization of PAP (green) and GAD65/67 (red) was seen in several areas of brain.

<p>In cerebral Purkinje cells (A–C), strong colocalization was seen especially in the axon hillock of the neuron (small picture in C; yellow color and white arrows depicting the colocalization). Similarly, PAP was present in GABAergic neurons in prefrontal cortex (PFC; infralimbic cortex) (D–F). Scale bars are 10 µm.</p