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Pericyte-specific expression of Rgs5: implications for PDGF and EDG receptor signaling during vascular maturation¹

HYESEON CHO, TOHRU KOZASA^*, CECILIA BONDJERS, CHRISTER BETSHOLTZ and JOHN H. KEHRL²

B-cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA;
^* Department of Pharmacology, University of Illinois, Chicago, Illinois, USA; and
Department of Medical Biochemistry, University of Goteborg, Goteborg, Sweden

²Correspondence: NIAID, Bldg. 10, Rm. 11B-13, 10 Center Dr., MSC 1876, Bethesda, MD 20892-1876, USA. E-mail: jkehrl{at}niaid.nih.gov

SPECIFIC AIMS

RGS proteins finely tune heterotrimeric G-protein signaling by acting as GTPase-activating proteins for G subunits. This study addresses the potential importance of Rgs5 in the regulation of PDGF and EDG receptor signaling during vascular maturation.

PRINCIPAL FINDINGS

1. Rgs5 exhibits a striking and extensive expression in the arterial walls of E12.5 to E17.5 mouse embryos
We analyzed developing mouse embryos by in situ hybridization using sense and antisense Rgs5 RNA probes. E8.5 and E10.5 embryos had no detectable hybridization signal although a strong signal overlaid extra embryonic tissues. E12.5 embryos had a moderate signal in the region of the perineural vascular plexa and aorta. Two days later in development, a striking signal appeared in most major organs with the exception of the liver and lung. The aorta and major vessels of the E14.5 mice strongly expressed Rgs5. E15.5, E17.5, and newborn mice had similar patterns of Rgs5 expression although the overall level of expression progressively declined.

Higher power photomicrographs of blood vessels from E11.5 and E12.5 embryos demonstrated Rgs5 expression in the aortic sac and major vessels and 1 day later in the developing aorta. The Rgs5 signal seemed to overlay the vSMC/pericyte layers rather than the endothelial cells. Aorta expression remained high for the remainder of development. The pulmonary artery had a moderate signal whereas the inferior and superior vena cava lacked one. A prominent increase in the amount of signal in the peripheral arteries occurred between days E13.5 and E15.5. Within the central nervous system (CNS), E12.5 embryos had a signal that overlaid the perineural plexa, but the rare penetrating vessel present contained very low levels of Rgs5. E14.5 and E17.5 embryos exhibited high levels of Rgs5 expression in their CNS, particularly associated with their numerous penetrating vessels. The choroid and hyaloid vascular plexa of E14.5 and later embryos expressed high levels of Rgs5. The developing kidney expressed Rgs5 with most of the signal localized to the renal arteries. In the adult, the major renal arteries, afferent and efferent renal arterioles, and the mesangial cells of the renal glomeruli expressed Rgs5.

2. Distribution and location of Rgs5-positive cells typified that of pericytes and overlap with the known expression pattern of platelet-derived growth factor B receptor (PDGF-Rß)
PDGF-Rß expression occurs in developing blood vessels in a pattern reminiscent of Rgs5. Several layers of PDGF-Rß-positive cells surround large arteries but not veins. A single layer covers smaller arteries and a noncontiguous layer overlays capillaries. Like Rgs5-positive cells, PDGFR-ß-positive cells are abundant in capillary plexa. Disruption of PDGF-B or PDGFR-ß in mice leads to lethal hemorrhage during embryonic development and the absence of kidney mesangial cells and microvascular pericytes. To assess the effect of blocking PDGF-B/PDGFR-ß signaling on Rgs5 expression, we compared E14.5 PDGF-B -/- and PDGF-Rß -/- mice to control mice. The brains of the PDGFR-ß -/- and PDGF-B -/- mice lacked the striking Rgs5 hybridization pattern seen in wild-type (Fig. 1 A–D). The mutant mice also exhibited a reduced Rgs5 expression in their hyaloid, choroid, and perineural plexa although a few Rgs5-positive cells persisted (Fig. 1E-J ). The PDGF-B mutants had reduced levels of Rgs5 in their small and medium-sized arteries; however, strong Rgs5 expression remained in the developing aorta and major renal arteries of these mice (Fig. 1K-P ). Thus, the loss of pericytes in the PDGF mutants results in the loss of Rgs5 expression from developing microvessels.

View larger version (160K): [in this window] [in a new window]   Figure 1. Composite of photomicrographs showing Rgs5 expression in blood vessels from E14.5 wild-type, E14.5 PDGF-B -/-, and E14.5 PDGFR-ß -/- mice. Photomicrographs showing emulsion autoradiograms of A) wild-type brain, x100 dark field, B) PDGFR-ß -/-, x100 dark field, C) wild-type CNS penetrating vessel, x1000, D) PDGF-B -/- CNS penetrating vessel, x1000, E, F) PDGF-B -/- choroids plexus, x200 dark and bright field, G, H) PDGF-B -/- and wild-type perineural plexus, respectively, x1000, I, J) wild-type and PDGFR-ß -/- hyaloid plexa, respectively, x100 dark field, K, L) wild-type and PDGFR-ß -/- kidney, respectively, x100 dark field, M, N) wild-type aorta, bright and dark fields, O, P) PDGF-B -/- aorta, bright and dark fields. Arrows indicate dense deposits of emulsion grains.

3. A potent GTPase-activating protein for Gi and Gq, RGS5, attenuates angiotensin II, endothelin-1-, S-1-P-, and PDGF-induced ERK-2 phosphorylation
We found that RGS5 enhanced the GTPase activity of Gi, Go, and Gq. We therefore examined the effect of RGS5 on Gi and/or Gq linked signaling pathways coupled to receptors that play important roles in vascular development and physiology. EDG-1 encodes a receptor for S-1-P and its disruption leads to a phenotype similar to that observed in the PDGF-B -/- mice. Cell migration toward PDGF appears to depend on EDG-1 expression. Therefore, we tested whether RGS5 affected ERK activation through EDG-1 or PDGF receptors. We found that RGS5 inhibited S-1-P- and PDGF-induced phosphorylation of ERK-2, suggesting that a PDGF-induced-ERK activation depended in part on the activation of heterotrimeric G-proteins (Fig. 2 ). We also noted that RGS5 attenuated ERK-2 phosphorylation induced by S-1-P, angiotensin II, or ET-1 using HASMC (Fig. 2) .

View larger version (42K): [in this window] [in a new window]   Figure 2. Inhibition of RGS5 on ERK activation induced by S-1-P, PDGF, angiotensin II, and ET-1. A) RGS5 inhibited S-1-P- and PDGF-induced ERK-2 phosphorylation in NIH-3T3. NIH-3T3 cells were transfected with constructs directing RGS5-HA, ERK-2-HA expression, and EDG-1 receptor. Cells were serum-starved and treated with S-1-P (100 nM) or PDGF (20 ng/mL) for 5 min. B)RGS5 inhibited S-1-P-induced ERK-2 phosphorylation via EDG-1 receptor in CHO cells. CHO cells w transfected with constructs directing expression of RGS5 (or RGS2), ERK-2, and EDG-1 receptor. Cells were serum-starved and treated with S-1-P (100 nM) or PMA (50 ng/mL) for 5 min. C)RGS5 attenuated angiotensin II-, ET-1-, or S-1-P-induced ERK-2 phosphorylation in HASMC. Cells were transfected with a construct expressing RGS5, serum-starved, and stimulated with angiotensin II (1 µM), endothelin-1 (100 nM), or S-1-P (100 nM) for 5 min. Protein lysates from harvested cells were used for immunoblotting with anti-phospho-ERK, anti-ERK, and anti-HA antibodies. Numbers in the RGS5 column indicate amounts of transfected RGS5 cDNA.

CONCLUSIONS AND SIGNIFICANCE

The major finding in the present study is developmentally regulated expression of Rgs5 in the fetal vasculature. Our data establish Rgs5 as a marker of developing pericytes and arterial smooth muscle cells. During embryogenesis, Rgs5 levels peak at E14.5 and decline thereafter, although considerable expression persists in the aorta, major vessels, and renal and cerebral microvasculature of adult animals. Besides expression in the developing microvasculature and major arteries, RGS5 acts as a Gq and Gi GAP and attenuates signaling through GPCRs important for vascular development such as EDG-1, AT-1, and ET-A receptors as well as through the PDGF receptor.

Since pericytes fail to enter the CNS along the angiogenic sprouts in the mice lacking PDGF-B or PDGFR-ß, the sharp reduction in Rgs5 expression in the brain parenchyma of these mice likely reflects the loss of pericytes. The persistence of pericytes and Rgs5-positive cells in the perineural vascular plexa of these mutant animals argues that PDGF-B/PDGF-Rß signaling is not the major inductive signal for Rgs5 expression in these cells.

RGS5 may function during the recruitment of pericyte/vSMC cells to endothelial tubes. PDGF-triggered cell migration depends in part on EDG-1 receptor signaling, presumably via activation of Gi and the release of Gß subunits. RGS5 as a GAP for Gi will shorten the duration that Gi remains GTP bound and encourage reformation of the heterodimer, limiting the free Gß subunits available for triggering cell migration. High levels of RGS proteins are known to significantly impair the migration of lymphocytes to chemokines; by analogy, the RGS5 present in pericytes/vSMC cells may impair their migration to chemoattractants such as PDGF or S-1-P. Therefore, one function of RGS5 may be to render cells no longer responsive to the migratory signals once they reach their final destination. However, complicating the analysis of Rgs5’s physiological role in developing vSMC/pericytes, its expression level within a cell may not reflect the functional status of the RGS5 protein. Its activity as a GAP and its intracellular location may both be regulated. In our experiments, the exposure of HASMC to S-1-P caused RGS5-GFP to localize to what appeared to be the leading edge of some of the cells. Although difficult to extrapolate this result to what happens during embryonic vascular maturation, it argues that RGS5 does not function at the trailing edge to prevent recruitment of the vSMC/pericyte away from the developing vessels by other chemoattractants. Studies of null mutants for Rgs5 will likely be needed to fully understand the functional role of Rgs5 in vivo.

In humans, macaque monkeys, and mice, high levels of RGS5 persist in the adult aorta and major arteries. Whereas RGS5 expression in the vSMC/pericytes may have a role in the migration of these cells during aorta development and remodeling, RGS5 likely serves some other function in the adult aorta and major arteries. RGS5 may regulate vascular smooth muscle proliferation and hypertrophy in response to angiotensin II or endothelins. Because arteries like the heart adapt to increased tension by thickening their walls, the level of RGS5 may alter the response of arteries to normal and pathological pressure changes. Finally, the finding that very high levels of RGS5 occur in certain highly vascular tumors provides a basis for exploring the role of RGS5 in the angiogenesis that accompanies progression and invasion of tumors.

View larger version (51K): [in this window] [in a new window]   Figure 3. Potential sites of action of RGS5 in PDGF/EDG-1 receptor-induced vascular maturation and possible consequences of RGS5 deficiency. Left: mesenchyme cells surrounding an endothelial tube differentiate to a pericyte/vSMC fate and assemble a vessel wall. Subsequent vessel enlargement and angiogenic sprouting depend on PDGF-B released from endothelial cells, which in part signals through EDG-1 receptors. RGS5 expression is indicated by gray coloration of the cells. 1, RGS5 could function during the initial migration of vSMC/pericytes to the endothelial tube, acting once the vSMC/pericyte has been recruited to the developing vessel. 2, RGS5 may limit vessel size by inhibiting PDGF-B signaling and decreasing cell proliferation or the recruitment of additional vSMC/pericytes. 3, RGS5 may function in directing the migration of vSMC/pericytes along angiogenic sprouts. Right: possible consequences of the loss of RGS5; cells lacking RGS5 indicated by their lack of gray coloration. 1, The loss of RGS5 could result in problems in the initial recruitment of vSMC/pericytes to vessel walls. Either excessive accumulation or a failure to retain cells might ensue. 2, Enhanced vessel wall thickness is possible due to excessive PDGFR-ß and EDG-1 receptor signaling. 3, The directed migration of vSMC/pericytes along angiogenic sprouts may be impaired due to excessive or inappropriate signaling through PDGFR-ß or EDG-1 receptors. Figure based in part on results from Hellstrom et al., Development. vol. 126, p. 3047, 1999.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0340fje; to cite this article, use FASEB J. (January 2, 2003) 10.1096/fj.02-0340fje

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