A role of the (pro)renin receptor in neuronal cell differentiation

Abstract

The (pro)renin receptor [(P)RR] plays a pivotal role in the renin-angiotensin system. Experimental models emphasize the role of (P)RR in organ damage associated with hypertension and diabetes. However, a mutation of the (P)RR gene, resulting in frame deletion of exon 4 [Delta4-(P)RR] is associated with X-linked mental retardation (XLMR) and epilepsy pointing to a novel role of (P)RR in brain development and cognitive function. We have studied (P)RR expression in mouse brain, as well as the effect of transfection of Delta4-(P)RR on neuronal differentiation of rat neuroendocrine PC-12 cells induced by nerve growth factor (NGF). In situ hybridization showed a wide distribution of (P)RR, including in key regions involved in the regulation of blood pressure and body fluid homeostasis. In mouse neurons, the receptor is on the plasma membrane and in synaptic vesicles, and stimulation by renin provokes ERK1/2 phosphorylation. In PC-12 cells, (P)RR localized mainly in the Golgi and in endoplasmic reticulum and redistributed to neurite projections during NGF-induced differentiation. In contrast, Delta4-(P)RR remained cytosolic and inhibited NGF-induced neuronal differentiation and ERK1/2 activation. Cotransfection of PC-12 cells with (P)RR and Delta4-(P)RR cDNA resulted in altered localization of (P)RR and inhibited (P)RR redistribution to neurite projections upon NGF stimulation. Furthermore, (P)RR dimerized with itself and with Delta4-(P)RR, suggesting that the XLMR and epilepsy phenotype resulted from a dominant-negative effect of Delta4-(P)RR, which coexists with normal transcript in affected males. In conclusion, our results show that (P)RR is expressed in mouse brain and suggest that the XLMR and epilepsy phenotype might result from a dominant-negative effect of the Delta4-(P)RR protein.

Fig. 1.

Expression of (P)RR in key brain regions involved in the regulation of blood pressure and body fluid homeostasis by in situ hybridization with radiolabeled (P)RR antisense riboprobes and counterstained with toluidine blue. (P)RR is highly expressed in neurons of the subfornical organ (SFO; A); the supraoptic nucleus (SO; B); the rostral ventrolateral medulla (RVLM; C); the paraventricular nuclei (PaV; D) and the nucleus tractus solitarius (NTS; E). Mve, medial vestibular nucleus; SpVe, spinal vestibular nucleus; vsc, ventral spinocerebellar tract; 3V, third ventricle.

Fig. 2.

Expression of (P)RR in adult mouse forebrain by in situ hybridization with radiolabeled riboprobes (A and B) Low-magnification bright field images of mouse brain sections counterstained with toluidine blue after ISH with (P)RR antisense probe (A) or sense probe (B). A strong expression of (P)RR mRNA was detected in the cortex, the thalamus, and the pyramidal cell layer of CA3. A sense probe did not display any specific signal. C–N: higher magnification images showing the hybridization signals obtained with the antisense probe in the cerebral cortex (C–F; 1, 2, 4, 6b refer to the cortical layers and WM referes to the white matter), the hippocampal formation (F–K; PL refers to pyramidal cell layer in CA1, CA2, and CA3 fields; GL refers to granule cell layer in the dentate gyrus); the thalamus (K, L; DLG and VPL refer to dorsal lateral geniculate nucleus and ventral posteromedial thalamic nucleus, respectively); and the choroid plexus (M; LV indicates the lateral ventricle). Background levels obtained with the sense probe hybridized to a serial section are shown in for the dentate gyrus (J) and for the cortex (N), white matter, and CA1 field. Scale bar shown in N corresponds to 1,000 μm (A and B) and 60 μm (C–N).

Fig. 3.

Staining of (P)RR in primary neurons. A–C: neurons fixed in paraformaldehyde and permeabilized by Triton-X100 were stained with purified IgG to (P)RR (ab1623 diluted 1/1,000). A: (P)RR staining shows a punctated pattern inside the neuronal body with reinforcement at the plasma membrane and along the neurites. B, C: localization of (P)RR in synaptic vesicle. B: Anti-(P)RR staining showed a punctuated labeling in neurons bodies and in neurites in primary neurons in culture. The double staining with monoclonal anti-synaptophysin that stains synaptic vesicles showed that (P)RR colocalized with synaptophysin. Higher magnification of vesicles in neurites (C) showed that not every synaptophysin-positive vesicle (green) was positive for (P)RR (red). Scales bars are 10 μm. D: higher magnification the colocalization of (P)RR with synatophysin in a synaptic vesicle.

Fig. 4.

Renin-induced phosphorylation of ERK1/2 on neurons and PC-12 cells. A: primary mouse neurons were cultured for 7 days and serum deprived overnight prior to stimulation. Renin (10 nM) induced ERK phosphorylation detectable at 10 min and lasted for at least 30 min. The graph summarizes the time course of ERK1/2 phosphorylation induced by renin in neurons; the errors bars represent the SE of 3 independent experiments performed in triplicate. B: PC-12 cells were serum starved and then stimulated with 10 nM renin or 10% serum. C: NGF-induced ERK1/2 activation is inhibited in PC-12 cells expressing Δ4-(P)RR. Cells were transfected with plasmids encoding (P)RR or Δ4-(P)RR and stimulated with serum or NGF. Cell lysates were analyzed by Western blot for ERK1/2 and phospho-ERK1/2. Data in this figure represent mean ± SD of 4 experiments and ***P < 0.001 by Student's t-test.

Fig. 5.

Cellular distribution of (P)RR and Δ4-(P)RR. Following transfection, the cells were treated without NGF (A) or with NGF (B) and subjected to immunofluorescence staining. V5-tagged proteins are stained with AlexaFluor-488 (green) and nuclei stained with Sytox-orange (red). Actin is stained with Phalloidin-633 (blue). Scale bar represents 10 μm. C: effects of cotransfection of (P)RR and Δ4(P)RR on cellular distribution of (P)RR. PC-12 cells were cotransfected with plasmids encoding (P)RR-GFP (green) and Δ4(P)RR-V5, cultured in the presence or absence of NGF, and subjected to immunofluorescence staining. Δ4(P)RR-V5 is stained with AlexaFluor-594 (red) and nuclei stained anti-lamin B and detected with Alexa Fluor-647 (blue). Scale bar represents 20 μm.

Fig. 6.

Dimerization of (P)RR and Δ4(P)RR. PC12 cells were transfected with plasmids encoding GFP or V5 tagged (P)RR or Δ4-(P)RR and cultured for 48 h. Coimmunoprecipitation of cell lysates was performed using anti-V5 antibody and Western blot analysis using anti-GFP antibody. Ly, cell lysate; IP, immunoprecipitates.