Neuropilins 1 and 2 (NRP1 and NRP2) are 130-kDa transmembrane receptors expressed on a range of cell types that mediate the effects of two independent ligand families: class 3 semaphorins (SEMA3) and members of the vascular endothelial growth factor (VEGF) family. Interaction of neuropilins with SEMA3 family members regulates axonal guidance in the central and peripheral nervous systems (reviewed by Bagri et al
1- Bagri A.
- Tessier-Lavigne M.
Neuropilins as Semaphorin receptors: in vivo functions in neuronal cell migration and axon guidance.
). Neuropilins have been shown to regulate angiogenesis by acting as coreceptors for VEGF on endothelial cells. Although both neuropilins bind SEMA3 proteins, they display distinctive ligand binding preferences. SEMA3A binds preferentially to NRP1, whereas SEMA3F binds with high affinity to NRP2. Similarly, the VEGF binding profiles of NRP1 and NRP2 are distinct: whereas both receptors bind VEGF-A, only NRP2 binds VEGF-C and VEGF-D (reviewed by Bielenberg and Klagsbrun
2- Bielenberg D.R.
- Klagsbrun M.
Targeting endothelial and tumor cells with semaphorins.
). Members of the plexin family, in particular plexins A1 through A4, are also necessary for SEMA3-mediated neuropilin-dependent signaling. Binding of SEMA3 ligands to neuropilins initiates a cascade of signals leading to marked changes in cell shape, which in turn mediate repulsion of NRP-expressing cells from the SEMA3 source. Such alterations in cell phenotype underlie the well-characterized growth cone collapse that drives axon guidance,
3Neuropilin is a receptor for the axonal chemorepellent Semaphorin III.
, 4- Chen H.
- Chédotal A.
- He Z.
- Goodman C.S.
- Tessier-Lavigne M.
Neuropilin-2, a novel member of the neuropilin family, is a high affinity receptor for the semaphorins Sema E and Sema IV but not Sema III [Erratum appeared in Neuron 1997, 19:559].
as well as the antiangiogenic effects of SEMA3 ligands described previously.
5- Bielenberg D.R.
- Hida Y.
- Shimizu A.
- Kaipainen A.
- Kreuter M.
- Kim C.C.
- Klagsbrun M.
Semaphorin 3F, a chemorepulsant for endothelial cells, induces a poorly vascularized, encapsulated, nonmetastatic tumor phenotype.
, 6- Kigel B.
- Varshavsky A.
- Kessler O.
- Neufeld G.
Successful inhibition of tumor development by specific class-3 semaphorins is associated with expression of appropriate semaphorin receptors by tumor cells.
The expression patterns for NRP1 and NRP2 are largely nonoverlapping
in vivo, suggesting discrete functions for the two proteins. Transgenic overexpression of
Nrp1 resulted in embryonic lethality arising from excess and dilated vessels, hemorrhage, and malformation of the heart and limbs.
7- Kitsukawa T.
- Shimono A.
- Kawakami A.
- Kondoh H.
- Fujisawa H.
Overexpression of a membrane protein, neuropilin, in chimeric mice causes anomalies in the cardiovascular system, nervous system and limbs.
Conversely, genetic ablation strategies in mice and other experimental organisms revealed functions for
Nrp1 in neuron guidance and cardiovascular development.
8- Kawasaki T.
- Kitsukawa T.
- Bekku Y.
- Matsuda Y.
- Sanbo M.
- Yagi T.
- Fujisawa H.
A requirement for neuropilin-1 in embryonic vessel formation.
, 9- Lee P.
- Goishi K.
- Davidson A.J.
- Mannix R.
- Zon L.
- Klagsbrun M.
Neuropilin-1 is required for vascular development and is a mediator of VEGF-dependent angiogenesis in zebrafish.
, 10- Bron R.
- Eickholt B.J.
- Vermeren M.
- Fragale N.
- Cohen J.
Functional knockdown of neuropilin-1 in the developing chick nervous system by siRNA hairpins phenocopies genetic ablation in the mouse.
The demonstration that both overexpression and deletion of
Nrp1 evoke profound developmental abnormalities illustrates the requirement for
Nrp1 function during essential developmental processes, but also suggests that NRP1 activity is dose-dependent. Mice with targeted mutation of
Nrp2 survive to adulthood, but display aberrant development and/or organization of cranial and spinal nerves, as well as a profound decrease in lymphatic capillaries.
11- Giger R.J.
- Cloutier J.F.
- Sahay A.
- Prinjha R.K.
- Levengood D.V.
- Moore S.E.
- Pickering S.
- Simmons D.
- Rastan S.
- Walsh F.S.
- Kolodkin A.L.
- Ginty D.D.
- Geppert M.
Neuropilin-2 is required in vivo for selective axon guidance responses to secreted semaphorins.
, 12- Chen H.
- Bagri A.
- Zupicich J.A.
- Zou Y.
- Stoeckli E.
- Pleasure S.J.
- Lowenstein D.H.
- Skarnes W.C.
- Chédotal A.
- Tessier-Lavigne M.
Neuropilin-2 regulates the development of selective cranial and sensory nerves and hippocampal mossy fiber projections.
, 13- Walz A.
- Rodriguez I.
- Mombaerts P.
Aberrant sensory innervation of the olfactory bulb in neuropilin-2 mutant mice.
, 14- Yuan L.
- Moyon D.
- Pardanaud L.
- Bréant C.
- Karkkainen M.J.
- Alitalo K.
- Eichmann A.
Abnormal lymphatic vessel development in neuropilin 2 mutant mice.
Mice lacking both
Nrp1 and
Nrp2 die early in embryonic development (embryonic day 8.5) as a result of impaired angiogenesis of the yolk sac and other structures.
15- Takashima S.
- Kitakaze M.
- Asakura M.
- Asanuma H.
- Sanada S.
- Tashiro F.
- Niwa H.
- Miyazaki Ji J.
- Hirota S.
- Kitamura Y.
- Kitsukawa T.
- Fujisawa H.
- Klagsbrun M.
- Hori M.
Targeting of both mouse neuropilin-1 and neuropilin-2 genes severely impairs developmental yolk sac and embryonic angiogenesis.
Analyses of signaling pathways that underlie neuropilin-mediated effects, such as collapse and inhibition of migration, have identified Rho family G proteins as important regulatory nodes.
16- Fournier A.E.
- Nakamura F.
- Kawamoto S.
- Goshima Y.
- Kalb R.G.
- Strittmatter S.M.
Semaphorin3A enhances endocytosis at sites of receptor-F-actin colocalization during growth cone collapse.
, 17- Shimizu A.
- Mammoto A.
- Italiano Jr, J.E.
- Pravda E.
- Dudley A.C.
- Ingber D.E.
- Klagsbrun M.
ABL2/ARG tyrosine kinase mediates SEMA3F-induced RhoA inactivation and cytoskeleton collapse in human glioma cells.
, 18- Liu B.P.
- Strittmatter S.M.
Semaphorin-mediated axonal guidance via Rho-related G proteins.
We recently demonstrated that SEMA3F signals via a NRP2-plexin A1 complex to mediate cytoskeletal collapse in tumor and endothelial cells.
17- Shimizu A.
- Mammoto A.
- Italiano Jr, J.E.
- Pravda E.
- Dudley A.C.
- Ingber D.E.
- Klagsbrun M.
ABL2/ARG tyrosine kinase mediates SEMA3F-induced RhoA inactivation and cytoskeleton collapse in human glioma cells.
In that study, we identified ABL2/ARG and RhoA/Rho kinase (ROCK) as components of a signaling cascade leading to cofilin-mediated actin depolymerization and an ensuing reduction in cell contractility and migration. RhoA and ROCK are also known regulators of smooth muscle (SM) function, namely, the maintenance of tone and contractility in vessels and other hollow organs (reviewed by Puetz et al
19- Puetz S.
- Lubomirov L.T.
- Pfitzer G.
Regulation of smooth muscle contraction by small GTPases.
). Although several reports have described expression of NRP1 in vascular smooth muscle cells (SMCs) and have identified a role for NRP1 in regulation of SMC migration,
20- Ishida A.
- Murray J.
- Saito Y.
- Kanthou C.
- Benzakour O.
- Shibuya M.
- Wijelath E.S.
Expression of vascular endothelial growth factor receptors in smooth muscle cells.
, 21- Liu W.
- Parikh A.A.
- Stoeltzing O.
- Fan F.
- McCarty M.F.
- Wey J.
- Hicklin D.J.
- Ellis L.M.
Upregulation of neuropilin-1 by basic fibroblast growth factor enhances vascular smooth muscle cell migration in response to VEGF.
, 22- Banerjee S.
- Mehta S.
- Haque I.
- Sengupta K.
- Dhar K.
- Kambhampati S.
- Van Veldhuizen P.J.
- Banerjee S.K.
VEGF-A165 induces human aortic smooth muscle cell migration by activating neuropilin-1-VEGFR1-PI3K axis.
, 23- Pellet-Many C.
- Frankel P.
- Evans I.M.
- Herzog B.
- Jünemann-Ramírez M.
- Zachary I.C.
Neuropilin-1 mediates PDGF stimulation of vascular smooth muscle cell migration and signalling via p130Cas.
the function of neuropilins in SM
in vivo has been unexplored. SEMA3-dependent activation of neuropilins elicits effects on the cytoskeleton similar to those seen with inhibition of the Rho-ROCK axis. These observations imply that neuropilins are important regulators of the SM phenotype and provide a compelling rationale for determining their function in SM-rich hollow organs.
With the present study, we identify visceral SM as a major site of expression of Nrp2 in vivo. Alterations in SM phenotype contribute to various pathologies affecting hollow organs, such as fibrosis and obstruction. Using a combination of biochemical and functional evaluation of cytoskeletal integrity, together with mouse models of Nrp2 deficiency, we show that loss of Nrp2 in vivo enhances agonist-induced force generation in muscle strips and leads to major alterations in hollow organ function. Collectively, these findings implicate Nrp2 as a novel regulator of visceral SM contractility.
Materials and Methods
Ethics Statement
All animal studies were performed with approval from the Children's Hospital Boston Animal Care and Use Committee and with strict adherence to US Public Health Service and Office of Laboratory Animal Welfare guidelines.
Nrp2 Transgenic Mice
Nrp2+/LacZ mice were generated as described previously.
15- Takashima S.
- Kitakaze M.
- Asakura M.
- Asanuma H.
- Sanada S.
- Tashiro F.
- Niwa H.
- Miyazaki Ji J.
- Hirota S.
- Kitamura Y.
- Kitsukawa T.
- Fujisawa H.
- Klagsbrun M.
- Hori M.
Targeting of both mouse neuropilin-1 and neuropilin-2 genes severely impairs developmental yolk sac and embryonic angiogenesis.
Briefly, a targeting vector was constructed in which the translated portion of the first coding exon and the proximal part of the next intron of
Nrp2 were replaced with a promoterless
Escherichia coli β-galactosidase gene to produce
Nrp2+/LacZ mice. These mice were backcrossed to the C57BL/6 strain for >10 generations. Pups from heterozygous offspring mated to produce
Nrp2LacZ/LacZ (effectively,
Nrp2−/−) die soon after birth.
Nrp2+/gfp mice (also known as
Nrp2tm1.2Mom/MomJ) were purchased from the Jackson Laboratory (Bar Harbor, ME; stock no. 006700) and maintained in the C57BL/6 background.
Nrp2gfp/gfp mice were viable and fertile and were used for contractility experiments. SM-specific deletion of
Nrp2 after birth was accomplished by breeding mice harboring an inducible Cre recombinase (
CreERT2) under control of the SM22α promoter (hereafter referred to as
SM22α-CreERT2)
24- Kühbandner S.
- Brummer S.
- Metzger D.
- Chambon P.
- Hofmann F.
- Feil R.
Temporally controlled somatic mutagenesis in smooth muscle.
to those expressing a floxed allele of
Nrp2 (
Nrp2fl/fl), and treating offspring positive for both genotypes with 0.5 mg 4-hydroxytamoxifen (4-OHT) for 3 to 5 days. Tissues from mice were harvested for analysis at selected times after initiation of recombination.
Histological Analysis and IHC
After euthanasia, whole
Nrp2+/LacZ newborn pups (postnatal day P1) or organs (bladder, heart, and skeletal muscle) from
Nrp2+/LacZ mice (P28 or P56) were directly embedded in optimal cutting temperature compound (OCT: Sakura, Tokyo, Japan) and frozen with liquid nitrogen. For detection of β-galactosidase activity, cryosections (8 to 10 μm thick) were fixed in cold methanol for 10 minutes,
25- Bell P.
- Limberis M.
- Gao G.
- Wu D.
- Bove M.S.
- Sanmiguel J.C.
- Wilson J.M.
An optimized protocol for detection of E. coli beta-galactosidase in lung tissue following gene transfer.
rinsed with PBS (pH 7.2), and incubated overnight at 37°C in X-gal reagent [1 mg/mL 5-bromo-4-chloro-3-indolyl-β-
d-galactopyranoside diluted in dimethyl sulfoxide, 5 mmol/L K
3Fe(CN)
6, 5 mmol/L K
4Fe(CN)
6, and 2 mmol/L MgCl
2 in PBS (pH 6.5)]. The next day, sections were rinsed in PBS, counterstained with eosin Y solution alcoholic with phloxine (Sigma-Aldrich, St. Louis, MO), and mounted with Permount medium (Thermo Fisher Scientific, Waltham, MA). For IHC staining, cryosections were fixed in 2% paraformaldehyde in PBS for 10 minutes (for mNrp2 staining), rinsed with PBS, incubated in 3% H
2O
2 in methanol for 12 minutes to block endogenous peroxidases, rinsed with PBS, incubated in protein blocking solution (3% goat serum, 2% sheep serum in PBS) for 15 minutes, and then incubated overnight at 4°C in primary antibody (rabbit monoclonal (D39A5) anti-mouse Nrp2 IgG antibody (3366; Cell Signaling Technology, Danvers, MA) diluted in protein blocking solution. The next day, for Nrp2 staining, sections were rinsed with PBS, incubated for 1 hour in Alexa Fluor-488-conjugated goat anti-rabbit IgG (H+L) antibody (Invitrogen-Life Sciences, Carlsbad, CA) diluted in protein blocking solution, rinsed with PBS, counterstained with Hoechst 33258 dye (bisbenzimide; Sigma-Aldrich), and mounted with Fluoro-Gel medium (Electron Microscopy Sciences, Hatfield, PA).
For α-smooth muscle actin (α-SMA) IHC staining, cryosections were stained in a single-day procedure according to the manufacturer's instructions in a M.O.M. (Mouse-on-Mouse) basic immunodetection kit (BMK-2202; Vector Laboratories, Burlingame, CA) using mouse monoclonal anti-α-SMA primary antibody (no. M0851, clone 1A4; Dako, Carpinteria, CA). Sections were rinsed with PBS, incubated for 1 hour in M.O.M. biotinylated anti-mouse IgG (Vector Laboratories) diluted in protein blocking solution, rinsed with PBS, incubated in horseradish peroxidase-conjugated avidin (Vectastain Elite ABC kit, PK-6100; Vector Laboratories) for 30 minutes, washed with PBS, visualized with a 3,3′-diaminobenzidine peroxidase chromogenic substrate kit (SK-4100; Vector Laboratories), rinsed with water, counterstained with Gill no. 3 hematoxylin (Sigma-Aldrich), and mounted with Permount medium. Control specimens exposed to each secondary antibody alone showed no specific staining.
Photomicrographs were obtained using Optronics Engineering image analysis software version 1.0.0.4 (Bioscan, Seattle, WA) and Adobe Photoshop software version 5.0.2 installed in a PC microcomputer with a frame grabber and with a Sony 3-CCD camera mounted on a Nikon Eclipse E600 microscope. Scans of stained sections of newborn (postnatal day P1) mice were obtained using a Polaroid SprintScan 35 Plus microscope slide scanner (Meyer Instruments).
Cell Culture, Treatments, and Biochemical Analyses
Human primary SMC cultures obtained from bladder (HBSMC), colon (HCSMC), and bronchus (HBrSMC) were purchased from ScienCell (San Diego, CA) and propagated in smooth muscle cell medium (SMCM; ScienCell). Human primary vascular SMCs obtained from pulmonary artery (HPASMC), coronary artery (HCASMC), or umbilical artery (HUASMC) were purchased from Lonza (Walkersville, MD) and cultured in smooth muscle growth medium (SmGM-2; Lonza). Spontaneously immortalized porcine aortic endothelial (PAE) cells were provided by Dr. Lena Claesson-Welsh (Uppsala University, Uppsala, Sweden) and maintained in F12 Ham's medium (Invitrogen-Life Technologies) supplemented with 10% fetal bovine serum (FBS). PAE cells overexpressing NRP1 (PAE/NRP1) or NRP2 (PAE/NRP2) were used as controls and described previously.
5- Bielenberg D.R.
- Hida Y.
- Shimizu A.
- Kaipainen A.
- Kreuter M.
- Kim C.C.
- Klagsbrun M.
Semaphorin 3F, a chemorepulsant for endothelial cells, induces a poorly vascularized, encapsulated, nonmetastatic tumor phenotype.
, 26- Soker S.
- Takashima S.
- Miao H.Q.
- Neufeld G.
- Klagsbrun M.
Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor.
Human bladder urothelial cells were propagated in ProstaLife medium (both from Lifeline Cell Technology, Frederick, MD), according to the manufacturer's guidelines.
For protein analysis, primary SMCs were seeded in six-well plates at a density of 10
5 cells/well in complete growth medium. For comparison of visceral and vascular SMC protein content, lysates were harvested when cells reached approximately 80% to 90% confluency in complete growth medium. For analysis of protein content after growth factor treatment, cells were switched after 24 hours in complete growth medium to serum-depleted medium for 24 hours, at which time growth factors were added to a final concentration of 1 nmol/L for a further 24 hours. At the end of the incubation period, cells were rinsed twice with ice-cold PBS and harvested in 1× lysis buffer for protein analysis as described previously.
27- Ramachandran A.
- Ranpura S.A.
- Gong E.M.
- Mulone M.
- Cannon Jr, G.M.
- Adam R.M.
An Akt- and Fra-1-dependent pathway mediates platelet-derived growth factor-induced expression of thrombomodulin, a novel regulator of smooth muscle cell migration.
In selected experiments, membrane-enriched fractions were prepared. Briefly, cells were rinsed twice with ice-cold PBS, scraped in PBS and 2 mmol/L EDTA, and pelleted at approximately 250 ×
g at 4°C. Cell pellets were resuspended in buffer M (50 mmol/L HEPES pH 7.4, 10 mmol/L NaCl, 5 mmol/L MgCl
2, and 0.1 mmol/L EDTA supplemented with protease inhibitors) and incubated for 10 minutes on ice, to promote cell swelling. Cells were disrupted mechanically using a Potter-Elvehjem homogenizer; the resulting homogenates were centrifuged at 16,000 ×
g for 10 minutes at 4°C, and pellets were resuspended in buffer M containing 1% Triton X-100 and incubated on ice for 30 minutes. Samples were centrifuged at 16,000 ×
g for 15 minutes at 4°C and supernatants were assayed for protein content. For comparison of protein content between various mouse organs, a wild-type C57BL/6 mouse (age P56) was euthanized, and organs were snap-frozen in liquid nitrogen. Frozen organs were ground to a powder, and protein lysates were isolated with radioimmunoprecipitation assay buffer (50 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS; Boston Bioproducts, Ashland, MA) supplemented with Complete Mini protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Protein concentrations were determined using a Bio-Rad (Hercules, CA) DC protein assay kit.
RhoA activity assays were performed using a RhoA activation assay kit based on rhotekin pull-down, according to the manufacturer's instructions (Cytoskeleton, Denver, CO) and as described previously.
17- Shimizu A.
- Mammoto A.
- Italiano Jr, J.E.
- Pravda E.
- Dudley A.C.
- Ingber D.E.
- Klagsbrun M.
ABL2/ARG tyrosine kinase mediates SEMA3F-induced RhoA inactivation and cytoskeleton collapse in human glioma cells.
Briefly, 1.5 × 10
6 cells HBSMCs were plated in full growth medium and allowed to adhere overnight. Next, cells were incubated with 320 ng/mL SEMA3F for 0 to 30 minutes, then were washed with PBS and lysed with cell lysis buffer (25 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 5 mmol/L MgCl
2, and 1% Triton X-100). Whole-cell lysates were incubated with rhotekin-bound beads for 1.5 hours at 4°C and washed with low-salt buffer (25 mmol/L Tris-HCl pH 7.5, 40 mmol/L NaCl, 15 mmol/L MgCl
2). Proteins were dissociated from beads by incubation in reducing sample buffer and were analyzed by Western blotting.
Western Blotting
Proteins were diluted in reducing sample buffer and resolved on 7.5% polyacrylamide gels or 4% to 20% gradient polyacrylamide gels. Proteins were transferred to nitrocellulose membranes and blocked with 5% nonfat milk in Tris-buffered saline/Tween (50 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, and 0.05% Tween-20) for 1 hour. For mouse proteins, the membrane was incubated overnight at 4°C with rabbit monoclonal (D39A5) anti-mouse Nrp2 IgG antibody (no. 3366; Cell Signaling Technology), or mouse monoclonal (6C5) anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) IgG1 antibody (no. MAB374; Millipore, Billerica, MA). For human proteins, the membrane was incubated overnight at 4°C with one of the following antibodies: mouse monoclonal (C-9) anti-human NRP2 IgG2b antibody (no. SC-13117; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal (A-12) anti-human NRP1 IgG1 antibody (SC-5307; Santa Cruz Biotechnology), rabbit anti-plexin A1 polyclonal antibody (3813; Cell Signaling Technology), rabbit monoclonal (67B9) anti-RhoA IgG antibody (2117; Cell Signaling Technology), mouse monoclonal (MY-21) anti-myosin light chain (MLC) IgM antibody (M4401; Sigma-Aldrich), rabbit anti-phospho-MLC (pSer19) polyclonal antibody (M6068; Sigma-Aldrich), mouse monoclonal (AC-15) anti-β-actin IgG1 antibody (A5441; Sigma-Aldrich), or anti-calnexin polyclonal antibody (Abcam, Cambridge, MA). Membranes were washed with Tris-buffered saline/Tween and incubated in the appropriate secondary antibody, either horseradish peroxidase-linked donkey anti-rabbit IgG whole antibody or horseradish peroxidase-conjugated sheep anti-mouse IgG whole antibody (both from GE Healthcare, Piscataway, NJ), for 1 hour. Membranes were washed, exposed to enhanced chemiluminescence (Pierce SuperSignal West Pico chemiluminescent substrate; Thermo Fisher Scientific, Rockford, IL) and signals were visualized after exposure of membranes to X-ray film.
RT-PCR
RNA was isolated from semiconfluent dishes of HBSMCs or human bladder urothelial cells using TRIzol reagent (Invitrogen-Life Technologies) or from SM and urothelial compartments acquired by laser capture microdissection from cryosections of bladders of C57BL/6 mice using Arcturus PicoPure RNA isolation reagent (Life Technologies). RNA was reverse-transcribed using a high-capacity cDNA synthesis kit (Applied Biosystems-Life Technologies, Foster City, CA), as described previously.
27- Ramachandran A.
- Ranpura S.A.
- Gong E.M.
- Mulone M.
- Cannon Jr, G.M.
- Adam R.M.
An Akt- and Fra-1-dependent pathway mediates platelet-derived growth factor-induced expression of thrombomodulin, a novel regulator of smooth muscle cell migration.
cDNAs were amplified using gene-specific primers for mouse and human neuropilin 2 and semaphorin 3F (SABiosciences-Qiagen, Valencia, CA), and relative mRNA levels were determined with normalization to GAPDH, as described previously.
27- Ramachandran A.
- Ranpura S.A.
- Gong E.M.
- Mulone M.
- Cannon Jr, G.M.
- Adam R.M.
An Akt- and Fra-1-dependent pathway mediates platelet-derived growth factor-induced expression of thrombomodulin, a novel regulator of smooth muscle cell migration.
Purification of SEMA3 Proteins
SEMA3F protein was purified as described previously.
28- Bielenberg D.R.
- Shimizu A.
- Klagsbrun M.
Semaphorin-induced cytoskeletal collapse and repulsion of endothelial cells.
Briefly, HEK293 cells were transfected with human
SEMA3F plasmid (provided by Dr. Marc Tessier-Lavigne, Rockefeller University) using FuGENE 6 transfection reagent (Roche Applied Science). After 16 hours, the medium was replaced with serum-free CD293 medium (Invitrogen-Life Technologies). Conditioned medium containing SEMA3F protein was collected after 48 hours, centrifuged, filtered, adjusted to pH 7.4, purified over a HiTrap high-performance nickel chelating column (GE Healthcare) using fast protein liquid chromatography, eluted with imidazole-containing buffer, and desalted using a PD-10 gel filtration column (GE Healthcare). The resulting proteins were diluted in PBS (pH 7.2), and protein concentrations were established using a Bio-Rad DC protein assay.
Cell Collapse Assay
The SEMA3-induced cytoskeletal collapse assay was modified from a previously reported assay
28- Bielenberg D.R.
- Shimizu A.
- Klagsbrun M.
Semaphorin-induced cytoskeletal collapse and repulsion of endothelial cells.
for use with HBSMCs or HCSMCs. Briefly, acid-washed glass coverslips (18 × 18 mm) were placed into six-well dishes. HBSMCs or HCSMCs were plated onto the coverslips in full growth medium. After 16 hours, purified SEMA3 proteins (320 ng/mL) were added to half of the wells and were incubated at 37°C. After 0 to 60 minutes, cells were fixed in 4% paraformaldehyde in PBS for 10 minutes, washed with PBS, permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 5 minutes, washed with PBS, stained with Alexa Fluor 488 phalloidin (1:250 dilution; Invitrogen-Life Technologies) for 1 hour, washed with PBS, counterstained with Hoechst 33258 dye, and mounted with Fluoro-Gel medium (Electron Microscopy Sciences). The actin cytoskeleton of the SMCs was visualized and imaged using a Leica TCS SP2 AOBS confocal laser scanning system attached to a Leica DM IRE2 inverted microscope (Leica Microsystems, Wetzlar, Germany) and equipped with a 63× oil objective (NA 1.4), a 1.6× optivar, a 488-nm argon ion laser (for F-actin), and a 405-nm diode (for nuclei). Differential interference contrast images were also obtained. Leica confocal software LCS and ImageJ software (NIH, Bethesda, MD) were used to scale recorded images. In selected experiments, cells were pretreated with 10 µmol/L Rho kinase inhibitor Y27632 (EMD Chemicals, Darmstadt, Germany), 1 µg/mL RhoA inhibitor C3 transferase (Cytoskeleton Inc.), or 1 nmol/L HB-EGF.
Optical Magnetic Twisting Cytometry
Primary HBSMCs were seeded in 96-well plates at a density of 10,000 cells/well; when confluent, cells were switched to serum-depleted medium 24 hours before analysis. On the day of measurement, cells were incubated with Arg-Gly-Asp (R-G-D)-coated beads (4.5 μm in diameter) for 20 minutes, to allow bead binding to integrins on the cell surface. Vehicle, SEMA3F (0.2 or 0.6 μg/mL), or Y27632 (10 μM) was then added at the indicated concentrations for an additional 40 minutes, after which plates were mounted on a microscope stage custom fitted for twisting cytometry.
29- Fabry B.
- Maksym G.N.
- Butler J.P.
- Glogauer M.
- Navajas D.
- Fredberg J.J.
Scaling the microrheology of living cells.
, 30- Trepat X.
- Deng L.
- An S.S.
- Navajas D.
- Tschumperlin D.J.
- Gerthoffer W.T.
- Butler J.P.
- Fredberg J.J.
Universal physical responses to stretch in the living cell.
, 31- Chen C.
- Krishnan R.
- Zhou E.
- Ramachandran A.
- Tambe D.
- Rajendran K.
- Adam R.M.
- Deng L.
- Fredberg J.J.
Fluidization and resolidification of the human bladder smooth muscle cell in response to transient stretch.
Briefly, beads were magnetized horizontally and then twisted vertically in an oscillatory magnetic field with a frequency of 0.75 Hz. From the ratio of the applied mechanical torque to the measured lateral bead displacement, we calculated the cell elastic modulus
G′, a measure of cell stiffness. For each experimental condition, data were pooled from thousands of beads attached to hundreds of cells from at least eight separate wells. Data are presented as the median
G′ in pascals per nanometer ± SEM.
Assessment of Bladder Function by Cystometry
To analyze bladder function in mice with constitutive knockout of
Nrp2, we performed conscious cystometry, essentially as described previously.
32- Thorneloe K.S.
- Meredith A.L.
- Knorn A.M.
- Aldrich R.W.
- Nelson M.T.
Urodynamic properties and neurotransmitter dependence of urinary bladder contractility in the BK channel deletion model of overactive bladder.
Briefly,
Nrp2-null mice and wild-type littermate controls were anesthetized with isoflurane and the bladder was exposed with a lower midline abdominal incision. A polyethylene catheter (PE10) was placed in the dome of the bladder and secured; the free end of the catheter was tunneled subcutaneously to the back of the mouse, where it was exteriorized. The abdominal incision was closed in two layers, and animals were allowed to recover. The end of the catheter was connected via a stopcock to an infusion pump (Harvard Apparatus, Holliston, MA) and physiological pressure transducer, and the bladder was filled with normal saline solution at a rate of 12.5 μL/minute. After an equilibration period of 20 to 30 minutes, intravesical pressure was monitored over multiple cycles of filling and voiding during a 60-minute time frame. From these data, parameters such as peak voiding pressure, intercontraction interval, and bladder compliance were determined.
Contractility Testing
Functional analysis of muscle strips was performed essentially as described previously.
33- Cristofaro V.
- Peters C.A.
- Yalla S.V.
- Sullivan M.P.
Smooth muscle caveolae differentially regulate specific agonist induced bladder contractions.
Briefly, bladders from female
Nrp2 wild-type and null mice were harvested with minimal handling into ice-cold Krebs solution (120 mmol/L NaCl, 5.9 mmol/L KCl, 25 mmol/L NaHCO
3, 1.2 mmol/L Na
2H
2PO
4, 1.2 mmol/L MgCl
2 · 6H
2O, 2.5 mmol/L CaCl
2, and 11.5 mmol/L dextrose). The bladders were incised longitudinally, and the mucosa was removed by dissection under a stereomicroscope. Tissue strips were attached to a force transducer (Grass Technologies, Quincy, MA) and were suspended in an organ bath maintained at 37°C and bubbled with a mixture of 95% O
2 and 5% CO
2. Bladder tissue was stretched to a force of 0.5 ×
g and equilibrated for 45 minutes. Contractile responses to 1 nmol/L to 10 µmol/L of the cholinergic agonist carbachol, 120 mmol/L KCl, and 10 µmol/L α-β-methyl-ATP and to electrical field stimulation (1 to 64 Hz, 40 V, 0.5-ms pulse width, 10-second duration) were measured in separate strips. Data were calculated as force (millinewtons) normalized by tissue cross-sectional area and are expressed as means ± SEM.
Statistical Analysis
Differences in contractile responses between Nrp2 wild-type and Nrp2gfp/gfp mice were determined by Student's t-test. P < 0.05 was considered significant. For analysis of cystometric data, a generalized estimating equation (GEE) model was used to analyze the differences between Nrp2gfp/gfp mice and Nrp2 wild-type mice and also to handle correlation of data obtained from the same mouse. Four voids were obtained per mouse, for a total of 16 voids in Nrp2gfp/gfp mice and 20 voids in Nrp2 wild-type mice.
Discussion
With this study, we provide the first demonstration of a role for Nrp2 in regulation of SM contractility. Although previous studies have reported expression of neuropilins in isolated vascular SMCs and have demonstrated expression of Nrp2 mRNA in the developing bladder,
4- Chen H.
- Chédotal A.
- He Z.
- Goodman C.S.
- Tessier-Lavigne M.
Neuropilin-2, a novel member of the neuropilin family, is a high affinity receptor for the semaphorins Sema E and Sema IV but not Sema III [Erratum appeared in Neuron 1997, 19:559].
none described a role for these proteins in SM
in vivo. Using a series of complementary mouse models, including those with constitutive or tissue-specific deficiency in Nrp2 expression, we show that the SM compartments of the bladder and gastrointestinal tract are among the sites with highest expression of Nrp2
in vivo. Loss of expression of Nrp2 in the bladder wall, either constitutively or in SM, led to profound functional changes, marked by enhanced force generation in response to a range of contractile agonists. In addition, we observed a significant effect on the gastrointestinal tract, characterized by alterations in intestinal transit and evidence of obstruction (data not shown).
Neuropilins are expressed in multiple cell types, including neurons, vascular cells, dendritic cells, epithelial cells, and SMCs. Several recent studies have described expression of both Nrp1 and Nrp2 in bladder epithelium and submucosa
in vivo and have demonstrated changes in their expression in response to inflammatory stimuli.
39- Saban M.R.
- Backer J.M.
- Backer M.V.
- Maier J.
- Fowler B.
- Davis C.A.
- Simpson C.
- Wu X.R.
- Birder L.
- Freeman M.R.
- Soker S.
- Hurst R.E.
- Saban R.
VEGF receptors and neuropilins are expressed in the urothelial and neuronal cells in normal mouse urinary bladder and are upregulated in inflammation.
, 40- Saban R.
- Saban M.R.
- Maier J.
- Fowler B.
- Tengowski M.
- Davis C.A.
- Wu X.R.
- Culkin D.J.
- Hauser P.
- Backer J.
- Hurst R.E.
Urothelial expression of neuropilins and VEGF receptors in control and interstitial cystitis patients.
, 41- Cheppudira B.P.
- Girard B.M.
- Malley S.E.
- Schutz K.C.
- May V.
- Vizzard M.A.
Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis.
, 42- Saban M.R.
- Sferra T.J.
- Davis C.A.
- Simpson C.
- Allen A.
- Maier J.
- Fowler B.
- Knowlton N.
- Birder L.
- Wu X.R.
- Saban R.
Neuropilin-VEGF signaling pathway acts as a key modulator of vascular, lymphatic, and inflammatory cell responses of the bladder to intravesical BCG treatment.
Our analysis of Nrp2 expression
in vivo using the
Nrp2+/LacZ and
Nrp2+/gfp reporter mice, together with immunofluorescence imaging of wild-type mouse tissues, revealed robust Nrp2 expression in SM but failed to detect significant Nrp2 expression in either bladder or gut epithelia. Expression of neuropilins in many cell types is typically down-regulated after birth, once the axonal guidance and vascular development functions have been fulfilled.
14- Yuan L.
- Moyon D.
- Pardanaud L.
- Bréant C.
- Karkkainen M.J.
- Alitalo K.
- Eichmann A.
Abnormal lymphatic vessel development in neuropilin 2 mutant mice.
, 34- Pozas E.
- Pascual M.
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- Guijarro P.
- Sotelo C.
- Chédotal A.
- Del Río J.A.
- Soriano E.
Age-dependent effects of secreted Semaphorins 3A, 3F, and 3E on developing hippocampal axons: in vitro effects and phenotype of Semaphorin 3A (−/−) mice.
However, we observed sustained expression of Nrp2 in visceral SM in the postnatal period, consistent with a function for Nrp2 in this location that is distinct from its neurovascular roles.
The function of hollow organs such as the bladder and gastrointestinal tract is to store waste products at low pressure and expel them under volitional control. Serious consequences can result from sustained increases in intraluminal pressure, especially in the case of the bladder, with initial hypertrophy of the bladder wall leading to fibrosis, transmission of elevated pressure to the kidneys and ensuing renal damage. Our initial findings with tissues from mice with constitutive knockout of Nrp2 revealed increased contractility of bladder muscle strips exposed to a range of contractile agonists, as well as alterations in voiding behavior in conscious mice. In particular, analysis of voiding function in conscious mice indicated a reduction in bladder compliance in
Nrp2-deficient animals, compared with controls. Decreased compliance in hollow organs can result from remodeling and thickening of SM in response to elevated pressures, as occurs secondary to bladder outlet obstruction, hypertension, or asthma, and is characterized by aberrant deposition and turnover of the extracellular matrix.
43The bladder extracellular matrix Part I: architecture, development and disease.
, 44- Brooke B.S.
- Karnik S.K.
- Li D.Y.
Extracellular matrix in vascular morphogenesis and disease: structure versus signal.
, 45Role of human airway smooth muscle in altered extracellular matrix production in asthma.
Although we cannot exclude the possibility, our observation that neither peak voiding pressures nor bladder weight in
Nrp2-null mice differed significantly from wild-type controls argues against the possibility that the reduced compliance observed in
Nrp2-null mice results from bladder outlet obstruction.
Because Nrp2 is expressed in multiple cell types of relevance to bladder wall contractility, including SMCs and neurons, initially it was not possible to ascribe the enhanced force generation in tissues lacking Nrp2 to a specific cell of origin. To address the contribution of SM-expressed Nrp2, we deleted the gene selectively in this tissue type, using SM22α to drive Cre recombinase. The demonstration that Nrp2 deficiency specifically in SM also led to enhanced contractility in muscle strips strongly supports a myogenic, as opposed to neurogenic, mechanism for increased force generation. Our analysis of laser capture-microdissected SM and urothelial compartments from mouse bladders, as well as analysis of human bladder SMCs and urothelial cells in culture, revealed robust expression of SEMA3F in epithelial cells, consistent with paracrine activation of NRP2 expressed on SMCs. It is important to note, however, that the tension testing analysis was conducted in the absence of exogenous SEMA3F or VEGF.
The observation that tissue strips from
Nrp2-null mice showed enhanced responses to contractile agonists suggests that Nrp2 may also interact functionally with canonical regulators of contractility, such as muscarinic and purinergic receptors and/or their effectors, independently of ligand. In support of this possibility, the cytoplasmic domain of both Nrp1 and Nrp2 harbors the amino acids Ser-Glu-Ala (S-E-A) at the C-terminus. This S-E-A motif possesses PDZ domain binding activity and has been shown to mediate interaction with the protein, neuropilin-interacting protein (NIP), also known as synectin or GIPC.
46Cloning and characterization of neuropilin-1-interacting protein: a PSD-95/Dlg/ZO-1 domain-containing protein that interacts with the cytoplasmic domain of neuropilin-1.
, 47- Gao Y.
- Li M.
- Chen W.
- Simons M.
Synectin, syndecan-4 cytoplasmic domain binding PDZ protein, inhibits cell migration.
GIPC in turn interacts with a number of proteins associated with the contractile apparatus, including transgelin/SM22α, which is highly enriched in SM.
48- Ewing R.M.
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- Duewel H.S.
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Large-scale mapping of human protein-protein interactions by mass spectrometry.
Notably, Nrp1, acting through GIPC, was found to elicit effects on endothelial cell spreading by modulating the internalization and adhesion function of α5β1 integrin.
49- Valdembri D.
- Caswell P.T.
- Anderson K.I.
- Schwarz J.P.
- König I.
- Astanina E.
- Caccavari F.
- Norman J.C.
- Humphries M.J.
- Bussolino F.
- Serini G.
Neuropilin-1/GIPC1 signaling regulates alpha5beta1 integrin traffic and function in endothelial cells.
Importantly, the ability of Nrp1 to affect α5β1 integrin occurred independently of interactions with SEMA3 ligand and of its function as a VEGF coreceptor. Taken together, these findings suggest that Nrp2 may regulate SM contractility through both ligand-dependent and ligand-independent processes.
Activation of cholinergic and purinergic receptors, as well as exposure of tissues to electrical field stimulation or depolarizing agents such as KCl, can evoke a range of different intracellular signals. All of them, however, are known to activate the RhoA-ROCK axis to promote SM contraction.
50- Janssen L.J.
- Tazzeo T.
- Zuo J.
- Pertens E.
- Keshavjee S.
KCl evokes contraction of airway smooth muscle via activation of RhoA and Rho-kinase.
, 51- Park S.Y.
- Song H.J.
- Sohn U.D.
Participation of Rho-associated kinase in electrical stimulated and acetylcholine-induced contraction of feline esophageal smooth muscle.
Stimuli transduced by neuropilin 2 also impinge on the RhoA-ROCK cascade. Data from primary culture SMCs (
Figure 3), as well as previous work from our research group,
17- Shimizu A.
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- Italiano Jr, J.E.
- Pravda E.
- Dudley A.C.
- Ingber D.E.
- Klagsbrun M.
ABL2/ARG tyrosine kinase mediates SEMA3F-induced RhoA inactivation and cytoskeleton collapse in human glioma cells.
showed that SEMA3F-mediated signaling via NRP2 inhibited RhoA activity. In SMCs, this led to decreased phosphorylation of MLC and a reduction in cytoskeletal stiffness as measured by optical magnetic twisting cytometry, consistent with stimulation of relaxation. In contrast, inhibition of SEMA3F function
in vivo by genetic ablation of Nrp2 resulted in loss of relaxation (ie, increased contractility), compared with tissues from controls with intact Nrp2.
Together, these observations are consistent with a model in which Nrp2, through binding of SEMA3F, maintains appropriate SM tone, and loss of Nrp2 enhances agonist-induced SM contractility by relieving inhibition on RhoA/ROCK signaling.
In summary, we provide the first evidence to support a role for Nrp2 in regulating contractility of visceral SM. The ability of Nrp2 to promote SM relaxation, along with its robust expression in hollow organs such as the bladder and intestine, implies that this axis could be exploited for therapeutic benefit in conditions characterized by inappropriate SM contractility. These include bladder and bowel hyperreflexia secondary to spinal cord injury, as well as gastrointestinal tract motility disorders such as intestinal pseudo-obstruction and paralytic ileus. The existence of a natural ligand for Nrp2, as well as pharmacological agents that target components of the Nrp2 signaling axis, suggests the potential for relatively rapid translation of this novel finding to the clinical setting.
Article info
Publication history
Published online: June 11, 2012
Accepted:
April 5,
2012
Copyright
© 2012 American Society for Investigative Pathology. Published by Elsevier Inc.