The FASEB Journal express article 10.1096/fj.02-0340fje. Published online January 2, 2003.

Pericyte-specific expression of Rgs5: implications for PDGF and EDG receptor signaling during vascular maturation Hyeseon Cho,* Tohru Kozasa,� Cecilia Bondjers,� Christer Betsholtz,� and John H. Kehrl*

*B-cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-1876; �Department of Pharmacology, University of Illinois, Chicago, Illinois; and �Department of Medical Biochemistry, University of Goteborg, Goteborg, Sweden

Corresponding author: John H. Kehrl, NIAID, Bldg.10, Rm. 11B-13, 10 Center Dr., MSC 1876, Bethesda, MD 20892-1876. E-mail: jkehrl@niaid.nih.gov

ABSTRACT

RGS proteins finely tune heterotrimeric G-protein signaling. Implying the need for such fine-tuning in the developing vascular system, in situ hybridization revealed a striking and extensive expression pattern of Rgs5 in the arterial walls of E12.5�E17.5 mouse embryos. The distribution and location of the Rgs5-positive cells typified that of pericytes and strikingly overlapped the known expression pattern of platelet-derived growth factor receptor (PDGFR)-β. Both E14.5 PDGFR-β- and platelet-derived growth factor (PDGF)-B-deficient mice exhibited markedly reduced levels of Rgs5 in their vascular plexa and small arteries. This likely reflects the loss of pericytes in the mutant mice. RGS5 acts as a potent GTPase activating protein for Giα and Gqα and it attenuated angiotensin II-, endothelin-1-, sphingosine-1-phosphate-, and PDGF-induced ERK-2 phosphorylation. Together these results indicate that RGS5 exerts control over PDGFR-β and GPCR-mediated signaling pathways active during fetal vascular maturation.

Key words: RGS • G-protein signal transduction • blood vessel physiology

D evelopment of the vascular system involves two principal cell types, endothelial cells and vascular smooth muscle cells (vSMC)/pericytes. Most vessels begin as an endothelial tube, which subsequently acquires a vSMC/pericyte coating. The lack of pericytes and

the presence of microaneurysm formation in mouse embryos deficient in platelet-derived growth factor receptor (PDGFR)-β has provided compelling evidence that PDGFR-β signaling is necessary for the recruitment of vSMC/pericytes to developing blood vessels (1). Because sprouting endothelial cells express platelet-derived growth factor (PDGF)-B whereas developing pericytes express PDGFR-β, paracrine signaling likely regulates the proliferation and migration of pericytes during vascular maturation. Disruption of Edg-1 in mice, which encodes for one of the receptors for sphingosine-1-phosphate (S-1-P), also causes intraembryonic hemorrhaging and intrauterine death because of a failure of vSMC/pericytes to migrate to arteries and capillaries to stabilize the developing blood vessels (2). Suggesting that EDG-1 receptor signaling acts downstream of PDGF signaling, cell migration toward PDGF apparently depends on EDG-1 expression (3, 4). PDGF signaling triggers the activation of sphingosine kinase, leading to the

accumulation of intracellular S-1-P and thus activation of EDG-1 as judged by the translocation of β-arrestin and phosphorylation of EDG-1 (3).

Besides the EDG-1 receptor other G protein-coupled receptors (GPCRs) such as the angiotensin II type 1 (AT-1) and type A endothelin (ET-A) receptors have important biological roles in the developing vascular system. For example, mice lacking AT-1 receptor isoforms show reduced postnatal survival, low baseline blood pressure, renal arteriolar thickening, and marked hypoplasia of the renal papilla (5). Other gene targeting experiments in mice have shown that endothelin-1 (ET-1)/ET-A-mediated signaling plays an essential role in the aortic arch patterning during mouse embryonic development (6).

Activated GPCRs such as EDG-1, ET-A, and AT-1 function as guanine nucleotide exchange factors for hetreotrimeric G proteins, which act as molecular switches to trigger downstream signaling pathways, producing the biological responses attributed to these receptors. The heterotrimeric G proteins are themselves subject to control by a family of proteins termed regulators of G-protein signaling (RGS). The RGS family consists of more than 25 protein members, and they negatively regulate GPCR-mediated signaling pathways. They act as GTPase activating proteins (GAPs) for Gα subunits, which limits the duration that a Gα subunit remains GTP bound (7). They can also function as effector antagonists blocking the interaction of an activated Gα subunit with its downstream effectors (7). Finally, they can directly inhibit some of the downstream effectors of Gα subunits such as adenylyl cyclases (8). All of the RGS family members contain an essential 120-amino acid domain called the RGS domain, and they participate in a wide variety of organismal functions, including olfaction, vision, cell migration, T-cell activation, synapse development in the hippocampus, and hypertrophic responses by the heart (8�12).

One of the members of this gene family, Rgs5, was first isolated by degenerate polymerase chain reaction (PCR) cloning from the mouse (13). High levels of Rgs5 expression have been detected in heart, lung, skeletal muscle, and small intestine; lower levels have been found in brain, placenta, liver, colon, and leukocytes (14). In this study, we show that much of the widespread expression of Rgs5 reflects blood vessel expression. Furthermore, the fetal vasculature expressed strikingly high amounts of Rgs5 in a pattern reminiscent of the PDGFR-β. Found in vSMC, RGS5 interfered with signaling through several GPCRs known for their importance in cardiovascular function as well as through the PDGFR. We discuss the possible role of high levels of RGS5 in developing vSMC/pericyte.

MATERIALS AND METHODS

Cell culture, transfection, and reagents

Proliferating human aortic smooth muscle cells (HASMC) were purchased from Clonetics (Walkersville, MD) and grown as suggested by the manufacturer. NIH-3T3 and Chinese hamster ovary (CHO) cells were grown in Dulbecco�s modified Eagle�s medium containing 10% fetal calf serum. NIH-3T3 cells (Qiagen, Valencia, CA) were transfected using Superfect. To transfect CHO cells and HASMC, we used �nucleofection,� a newly developed technology by Amaxa Biosystems (Cologne, Germany). We achieved ~20�40% transfection efficiency as assessed by the expression of various RGS-GFP fusion proteins, using a fluorescence-activated cell sorter

Calibur flow cytometer. The total amount of plasmid DNA for each transfection was always normalized with empty vector DNA. Angiotensin II, S-1-P, ET-1, PDGF, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma (St. Louis, MO) and Calbiochem (San Diego, CA), respectively.

In situ hybridization

An expressed sequence tag (Incyte Genomics, St. Louis, MO) for Rgs5 was identified and subcloned into a pBluescript vector. Antisense and sense riboprobes used for in situ hybridization were transcribed with T3 and T7 RNA polymerases in the presence of [35S]UTP (Amersham, Piscatway, NJ). Mouse embryo sections of various developmental ages and sections of adult mouse organs were prepared at Molecular Histology Laboratories (Gaithersburg, MD). In situ hybridization was performed as described previously (15). In all the experiments shown, adjacent sections were analyzed with either the antisense Rgs5 or the sense Rgs5 probe. No specific signal was observed with the sense probe.

GAP assays

To generate hexa-histidine tagged RGS5, a cDNA fragment encoding a full-length human RGS5 was subcloned into NdeI and BamHI restriction sites of pET15b vector (Novagen, Madison, WI). The production of recombinant his-tagged RGS5 in Escherichia coli, its purification, and the measurements of single-cycle GTPase rates were performed as described previously (16). Hydrolysis of [γ32-P]GTP (5�10 µM, Amersham) was measured in the absence or presence of 200 nM of His6RGS5, 100 nM of His6RGS4, or 20 nM of p115RhoGEF. For Gqα GAP assay, a mutant of Gqα, Gqα R183C, was used (16).

GPCR and PDGF signaling assays

To determine the effect of RGS5 in various GPCR- or PDFGR-mediated signaling pathways, NIH-3T3, CHO cells, or primary HASMC were transfected with a construct directing the expression of HA-tagged RGS5 (or RGS2) in the presence or absence of a construct directing the expression of ERK-2-HA. The EDG-1 receptor construct was obtained from Dr. T. Hla (University of Connecticut, Farmington, CT). After transfection, cells were serum-starved overnight and stimulated with various stimuli for 5 min. Then the cells were washed twice in cold PBS and lysed in kinase lysis buffer (20 mM HEPES, 2 mM EGTA, 50 mM β-glycerophosphate, 1 mM Na3VO4, 1% Triton-X100, 10% glycerol, 100 mM NaCl, 10 mM NaF, and protease inhibitor tablets [Boehringer Mannheim, Indianapolis, IN]). The resulting lysates were analyzed by immunoblotting with anti-phosphoERK monoclonal antibody (New England BioLabs, Beverly, MA), anti-ERK antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-HA monoclonal antibody (Covance, Richmond, CA).

Confocal microscopy

Images of HASMC transfected with RGS5-GFP fusion protein were collected on a Leica TCS-SP2 confocal microscope (Leica Microsystems, Exton, PA), using a 40× oil immersion objective (NA 1.32, confocal zoom 1). Fluorochromes were excited using an argon laser at 488 nm. Differential interference contrast images were collected simultaneously with the fluorescence

images, using the transmitted light detector. The confocal pinhole was set to 5 Airy disks. Z stacks of images were collected at a step size of 2.8 µm and later flattened to produce maximum projections. Time-lapse recording of the confocal images was performed for 40 min with 20-s intervals between collections. Images were processed using Leica TCS-SP2 software (version 2.770), Imaris 3.2 (Bitplane AG, Zurich Switzerland), and Adobe Photoshop 5.5 (Adobe Systems, San Jose, CA)

RESULTS

To examine the expression of Rgs5 during embryonic development, we analyzed developing mouse embryos by in situ hybridization, using sense and antisense Rgs5 RNA probes. Dark-field and corresponding bright-field photomicrographs photographed at 4�5× are shown (Fig. 1). E8.5 and E10.5 embryos had no detectable hybridization signal, although a strong signal overlaid extra-embryonic tissues (Fig. 1A and 1B). E12.5 embryos had a moderate signal in the region of the perineural vascular plexa (Fig. 1C and 1D) and aorta (not visible in section shown). Two days later in development, a striking and extensive hybridization appeared in most major organs, with the exception of the liver and lung. The aorta and major vessels of the E14.5 mice had strong hybridization signal associated with them (Fig. 1E and 1F). E15.5 (Fig. 1G and 1H), E17.5 (Fig. 1I and 1K) and newborn mice (data not shown) had similar patterns of Rgs5 expression, although the overall level of the hybridization signal progressively declined in the older mice. In contrast, E17.5 embryos (Fig. 1J) and the other embryos (data not shown) lacked a detectable hybridization signal when we used the Rgs5 sense probe.

Higher power photomicrographs of blood vessels from E11.5 and E12.5 embryos demonstrated a detectable Rgs5 signal in the aortic sac and major vessels (Fig. 2A) and 1 day later a strong signal in the region of the developing aorta (Fig. 2B). Whereas the Rgs5 signal appeared to overlay the vSMC/pericyte layers rather than the endothelial cells, resolving the specific cell type responsible for the signal origin is difficult. Aorta expression remained high for the remainder of development (Fig. 2C). The pulmonary artery also had signal, however less than did the aorta (Fig. 2D), and the inferior and superior vena cava lacked detectable signal (data not shown). The examination of the peripheral arteries of E13.5 and E15.5 embryos revealed a prominent increase in Rgs5 expression with age without significant expression in developing veins (Fig. 3E and 3F). Within the central nervous system (CNS), E12.5 embryos had signal that overlaid the perineural plexa; however, the rare penetrating vessel present contained very low levels (Fig. 2G and 2H). Older embryos, E14.5 and E17.5 (Fig. 2I�2L), exhibited high levels of Rgs5 in their CNS, including in association with their more numerous penetrating vessels.

The striking difference in hybridization signal between the CNS perineural plexa of E12.5 and E14.5 mice is shown (Fig. 3A and 3B). Both the choroid and hyaloid vascular plexa of E14.5 and later embryos contained high levels of Rgs5 expression (Fig. 3C�3E). The developing kidney also displayed prominent Rgs5 expression, with the majority of the signal localized to the renal arteries (Fig. 3F and 3G). In the adult, the major renal arteries, afferent and efferent renal arterioles, and the mesangial cells of the renal glomeruli expressed Rgs5 (Fig. 3H and 3I). Whereas adult rat cardiomyocytes have been reported to express Rgs5, the majority of Rgs5 signal in the developing mouse heart was localized to blood vessels. An E17.5 embryo heart section (Fig. 3J) showed prominent grains along the edge, and a higher magnification documented arteriolar expression (data not shown). Newborn mice prominently expressed Rgs5

in their bone marrow vessels (Fig. 3K). We also observed expression of Rgs5 in lung mesenchymal cells likely associated with developing bronchial smooth muscle cells (data not shown) as well as in gut mesenchymal cells of E14.5 mice and E17.5 (Fig. 1E and I). However, in the adult intestine, we observed signal only in small blood vessels of the intestinal vili (Fig. 3L). These last results suggest a temporally regulated Rgs5 expression in visceral smooth muscle cells from endodermally derived organs such as gut and lung.

PDGFR-β mRNA expression occurs in developing blood vessels in a pattern reminiscent of Rgs5. Several layers of PDGFR-β-positive cells surround large arteries but not veins. In addition, a single layer covers smaller arteries and a noncontiguous layer overlays capillaries. Like Rgs5-positive cells, PDGFR-β-positive cells are abundant in capillary plexa. Disruption of either PDGF-B or PDGFR-β in mice leads to lethal hemorrhage during embryonic development and the absence of kidney mesangial cells and microvascular pericytes (1, 17). To assess the effect of blocking PDGF-B/PDGFR-β signaling on Rgs5 expression, we compared E14.5 PDGF-B −/− and PDGFR-β −/− mice to control mice. The brains of the PDGFR-β −/− and PDGF-B −/− mice lacked the striking Rgs5 expression seen in the wild type (Fig. 4A�4D). The mutant mice also exhibited a reduced expression in their hyaloid, choroid, and perineural plexa, although importantly, an occasional Rgs5-positive cell persisted (Fig. 4E�4J). The PDGF-B −/− mice had reduced levels of Rgs5 in their small- and medium-sized arteries; however, strong Rgs5 expression persisted in the developing aortas and major renal arteries of these mice (Fig. 4 K�4P). Thus, the loss of pericytes in the PDGF mutants results in the loss of Rgs5 mRNA expression from developing microvessels.

To determine G protein specificities of human RGS5 and thus the types of receptors likely to be inhibited by RGS5, we examined GAP activities of RGS5 toward various Gα subunits. In vitro single turnover GTPase assays were performed with purified recombinant Gα proteins and recombinant RGS5. We found that RGS5 enhanced the GTPase activity of Giα as described previously (18) as well as that of Goα and Gqα (Fig. 5) nearly as efficiently as did RGS4 (19). However, the intrinsic GTPase activities of Gsα and G12α were unaltered by RGS5 (Fig. 5). Thus, the GAP activity of RGS5 is restricted to Giα and Gqα subfamilies.

Finally, we 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. We first determined whether RGS5 affected ERK activation through either the EDG-1 receptor or PDGFR. We transfected NIH-3T3 cells with expression vectors for HA-ERK2 and the EDG-1 receptor in the presence or absence of RGS5, exposed the cells to S-1-P for 5 min, and examined ERK-2 activation by immunoblotting with a phospho-ERK-specific antibody (Fig. 6A). Coexpression of RGS5 markedly attenuated S-1-P-induced phosphorylation of ERK-2. In the next set of experiments, we used the endogenous PDGFR to activate ERK-2. RGS5 also reduced PDGF-induced ERK-2 phosphorylation, suggesting that a PDGF-induced ERK activation depended in part on the activation of heterotrimeric G proteins. Using EDG-1 receptor null CHO cells, we showed that RGS5 specifically inhibited EDG-1-mediated ERK activation (Fig. 6B). Expectedly, RGS5 did not attenuate PMA-induced ERK phosphorylation. In addition, another member of the RGS family, RGS2, known to be a potent GAP for Gqα, showed no effect on EDG-1-mediated phosphorylation of ERK-2 (Fig. 6B). We then examined the effect of RGS5 on angiotensin II-, ET-1-, or S-1-P-induced ERK activation, using HASMC (Fig. 6C). Constructs that direct the

expression of RGS5 attenuated ERK-2 phosphorylation induced by all three agonists. Thus, the level of RGS5 in vSMC/pericyte cells likely determines the responsiveness of those cells to signaling through the PDGF, EDG-1, ET-1, and angiotensin II receptors.

In an attempt to understand the role of RGS5 in the recruitment of vascular smooth muscle cells during vascular maturation, we examined the intracellular localization of RGS5 following the exposure of HASMC to either S-1-P or PDGF. HASMC cells transfected with a construct that expresses RGS5-GFP exhibited an even distribution of GFP expression throughout the cell. Time-lapse recording of confocal images of live HASMC stimulated with S-1-P revealed that compared with nontransfected cells, which moved in response to S-1-P, RGS5-expressing cells appeared markedly less mobile (Fig. 7). Whereas S-1-P did not induce a marked change in the intracellular localization of RGS5-GFP, some RGS5-GFP-positive cells showed a polarized recruitment of RGS5-GFP to one side of the cell following S-1-P stimulation. Time-lapse recording of confocal images obtained following the exposure of HASMC to purified PDGF revealed substantial PDGF-induced membrane ruffling over the duration of the recording. Interesting, expression of RGS5-GFP, but not RGS1-GFP or RGS2-GFP, attenuated PDGF-induced membrane ruffling (data not shown). Thus, high levels of RGS5 not only inhibit PDGF-induced ERK activation, but also PDGF-induced membrane ruffling.

DISCUSSION

The major finding in the present study is developmentally regulated expression of Rgs5 in the fetal vasculature. Our data establish Rgs5 as a specific marker of developing pericytes and arterial smooth muscle cells, although in developing endodermally derived organs such as gut and lung, Rgs5 expression appears to localize in visceral rather than vascular smooth cells. During embryogenesis, Rgs5 mRNA levels peak at E14.5 and decline thereafter, although considerable expression persists in the aorta, major vessels, and the renal and cerebral microvasculature of adult animals. Besides the prominent expression in the developing microvasculature and major arteries, RGS5 acts as a Gqα and Giα GAP and attenuates signaling through GPCRs important for vascular development such as the EDG-1, AT-1, and ET-A receptors as well as through the PDGFR.

Does PDGFR-β signaling induce Rgs5 expression in developing pericytes? Because pericytes fail to enter the CNS along the angiogenic sprouts in the mice lacking PDGF-B or PDGFR-β, the sharp reduction of 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/PDGFR-β signaling is not a major inductive signal for Rgs5 expression in these cells. During vSMC/pericyte development, newly formed vessels likely release a signal that leads to generation of PDGFR-β-positive vSMC/pericyte progenitors from surrounding undifferentiated mesenchyme (20). Subsequently, PDGF-B released by the endothelial tube causes the proliferation and recruitment of these PDGFR-β-positive progenitors. The same inductive signal that leads to vSMC/pericyte fate may turn on Rgs5 expression.

The major question raised by this study is why vSMC/pericyte cells express such high levels of Rgs5 during vascular maturation. One attractive hypothesis is that RGS5 functions during the recruitment of vSMC/pericyte cells to endothelial tubes. PDGF-triggered cell migration depends in part on EDG-1 receptor signaling, presumably via the activation of Giα and the release of Gβγ

subunits. RGS5 by acting as a GAP for Giα will shorten the duration that Giα remains GTP bound and encourage the reformation of the heterotrimer, thus limiting the free Gβγ subunits available to trigger cell migration. High levels of RGS proteins are known to significantly impair the migration of lymphocytes to chemokines (10, 21), and by analogy, the RGS5 present in vSMC/pericyte 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 have reached their final destination. However, complicating the analysis of RGS5�s physiologic role in developing vSMC/pericytes, its expression level within a cell may not reflect the functional status of the RGS5 protein. Numerous other regulatory influences may alter its GAP activity or its intracellular localization. For example, the phosphorylation status of an RGS protein may modulate its GAP activity (22, 23). PDGF signaling could induce RGS5 phosphorylation, thereby either augmenting or inhibiting its effects on GPCR signaling. Alternatively, PDGFR and/or EDG receptor signaling might localize RGS5 to the plasma membrane, where it can function to fine tune GPCR signaling or to a subcellular location where it cannot access Gα subunits. Several RGS proteins translocate to the cell membrane upon GPCR signaling (16, 24). 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 it is 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 the 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/Rgs5 persist in the adult aorta. A comparison of aorta and vena cava medial mRNA expression in adult macaque monkeys, using a cDNA array containing 4048 human genes, has revealed RGS5 to be the most differentially expressed (25). Subsequent Northern blot and in situ hybridization studies have confirmed that RGS5 is highly expressed in adult human and macque aorta. 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. Because RGS5 attenuates signaling through the AT-1 and ET-A receptor, RGS5 may assist in the control of vascular smooth muscle proliferation and hypertrophy in response to angiotensin II or endothelins. The recent demonstration of a receptor-selective effect of RGS5 on AT-1 receptor-mediated signaling further supports a function role for RGS5 in the vascular system. A synthetic ribozyme targeted to RGS5 enhanced angiotensin II-induced MAPK activation in rat vascular smooth muscle cells (26). Finally, RGS5 may also be involved in the adaptation of vessels to normal and pathological pressure changes, because arteries like the heart adapt to increased tension by thickening their walls. RGS4 has been proposed to have such a function in the heart (12).

In summary, we show that Rgs5, a negative regulator of EDG-1, AT-1, and ET-A receptor-mediated signaling pathways, exhibits a developmentally regulated vascular expression pattern in which peak expression occurs at approximately E14.5 of mouse development. The abundant expression of Rgs5 in developing vSMC/pericyte implies a functional role for Rgs5 during the proliferation and recruitment of these cells to endothelial tubes. The persistent expression in vascular plexa, aorta, and major vessels argues for a distinct role for Rgs5 in adults. Finally, our finding along with the observation that very high levels of RGS5 occur in certain highly vascular tumors (27) provides a basis for exploring the role of RGS5 in the angiogenesis that accompanies the progression and invasion of tumors.

ACKNOWLEDGMENTS

We thank Mary Rust for editorial assistance and Dr. Anthony S. Fauci for his support.

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Received June 13, 2002; accepted November 19, 2002.

Fig. 1

Figure 1. Composite of photomicrographs showing sagittal sections of mouse conceptuses (E8 and E10) and embryos (E12 –E17.5). Dark field and corresponding bright field photomicrographs of hematoxylin-stained emulsion autoradiograms were obtained from in situ hybridization with 35S-labeled Rgs5 antisense riboprobe. E8.5 and E10.5 (A, B), E12.5 (C, D), E14.5 (E, F), E15.5 (G, H), and E17.5 (I, K) mice. Adjacent sections were hybridized with sense probe as controls for nonspecific background, and the result with E17.5 is shown in J. Magnification, ×4–5, all panels. A, aorta; Gu, gut; H, heart; K, kidney; Li, liver; Lu, lung; and U, uterus.

Fig. 2

Figure 2. Composite of photomicrographs showing sections of developing aorta and blood vessels. Photomicrographs of hematoxylin-stained emulsion autoradiograms showing Rgs5 expression in E11.5 aorta, ×1000 (A); E12.5 aorta, ×1000 (B); newborn aorta, ×400 (C); E17.5 pulmonary artery, ×400 (D); E13.5 peripheral artery, ×1000 (E); E15.5 peripheral artery, ×1000 (F); dark field of E12.5 brain, ×50 (G); E12.5 central nervous system (CNS) penetrating vessel, ×1000 (H); dark field of E14.5 brain, ×50 (I); E14.5 CNS penetrating vessel, ×1000 (J); dark field view of E17.5 brain, ×50 (K); and E17.5 penetrating vessel, ×1000 (L). Arrows point to emulsion grains in individual photomicrographs. V, vein.

Fig. 3

Figure 3. Composite of photomicrographs of vascular plexa, kidney, heart, bone marrow, and gut. Photomicrographs of hematoxylin-stained emulsion autoradiograms showing Rgs5 expression in E12.5 perineural plexus, ×1000 (A); E14.5 perineural plexus, ×1000 (B); E17.5 choroid plexus, ×1000 (C); newborn hyaloid plexus, ×100 dark field (D); newborn hyaloid plexus, ×1000 (E); E14.5 kidney, arrow points to renal artery, ×10 dark field (F); E17.5 kidney, arrow points to renal artery, ×10 dark field (G); adult kidney artery, ×100 (H); adult kidney glomerulus, ×1000 (I); E17.5 heart, ×5 dark field (J); new born bone marrow, ×1000 (K); and adult gut, ×1000 (L). The arrows indicate dense deposits of emulsion grains. A, aorta.

Fig. 4

Figure 4. Composite of photomicrographs showing sections of developing aorta and blood vessels from E14.5 wild-type, E14.5 PDGF B -/-, and E14.5 PDGFR-β -/- mice. Photomicrographs showing emulsion autoradiograms of wild-type brain, ×100 dark field (A); PDGFR-β −/−, ×100 dark field (B); wild-type CNS penetrating vessel, ×1000 (C); PDGF B −/− CNS penetrating vessel, ×1000 (D); PDGF B −/− choroids plexus, ×200 dark and bright field (E and F); PDGF B −/− and wild-type perineural plexus, respectively, ×1000 (G and H); wild-type and PDGFR-β −/− hyaloid plexa, respectively, ×100 dark field (I and J); wild-type and PDGFR-β −/− kidney, respectively, ×100 dark field (K and L); wild-type aorta, bright and dark fields (M and N); and PDGF B −/− aorta, bright and dark fields (O and P). The arrows indicate dense deposits of emulsion grains.

Fig. 5

Figure 5. Enhancement of GTPase activity of Giα and Gqα subfamilies by RGS5. The ability of RGS5 to accelerate the GTPase activities of Giα1 (A), Goα (B), GqαR183C (C), Gsα (D), and G12α (E) was measured. GTP hydrolysis reaction was started by addition of various Gα subunits loaded with [γ-32P]GTP to a reaction buffer in the absence or presence of RGS4 (100 nM), RGS5 (200 nM), or p115 RhoGEF (20 nM). “Basal” stands for the basal GTPase activity of each Gα subunit. Aliquots were removed from the reaction mixture at the specified intervals, and the amount of released 32Pi was measured by liquid scintillation spectrometry.

Fig. 6

Figure 6. 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 were 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 human aortic smooth muscle cells. 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. The numbers in the RGS5 column indicate amounts of transfected RGS5 cDNA. The data shown are representative of multiple experiments.

Fig. 7

Figure 7. Time-lapse confocal microscopy of live human aortic smooth muscle cells (HASMCs) stimulated with S-1-P. HASMCs were transfected with a construct expressing RGS5-GFP, serum-starved, and stimulated with S-1-P. Images were collected on a Leica TCS-SP2 confocal microscope, using a 40× oil immersion. The confocal pinhole was set to 5 Airy disks. Z stacks of images were collected at a step size of 2.8 µm and later flattened to produce maximum projections. Time-lapse recording of the confocal images was performed at 20-s intervals for 40 min. Four frames shown are the merged images of GFP and DIC before the addition of the agonist, and 10, 20, and 30 min after. S-1-P (600 nM in 0.1 ml) was added to cell chambers containing 0.5 ml of medium at the right lower corner. The white arrowhead indicates an RGS5-GFP-positive cell exhibiting polarized distribution of RGS5-GFP. The arrows indicate nontransfected HASMCs moving as compared with less mobile RGS5-GFP-positive cells.