ABSTRACT
Rap1 GTPase drives assembly of the Mig-10/RIAM/lamellipodin–Integrin–Talin (MIT) complex that enables integrin-dependent lymphocyte functions. Here we used tandem affinity tag-based proteomics to isolate and analyze the MIT complex and reveal that Phostensin (PTSN), a regulatory subunit of protein phosphatase 1, is a component of the complex. PTSN mediates de-phosphorylation of Rap1 thereby preserving the activity and membrane localization of Rap1 to stabilize the MIT complex. CRISPR/Cas9-induced deletion of PPP1R18, which encodes PTSN, markedly suppresses integrin activation in Jurkat human T cells. We generated apparently healthy Ppp1r18-/- mice that manifest lymphocytosis and reduced population of peripheral lymphoid tissues ascribable to defective activation of integrins αLβ2 and α4β7. Ppp1r18-/- T cells exhibit reduced capacity to induce colitis in a murine adoptive transfer model. Thus, PTSN enables lymphocyte integrin-mediated functions by dephosphorylating Rap1 to stabilize the MIT complex. As a consequence, loss of PTSN ameliorates T cell-mediated colitis.
SUMMARY Phostensin, a protein phosphatase 1 regulatory subunit, supports lymphocyte integrin-dependent functions by mediating dephosphorylation of Rap1 to stabilize the MIT complex thereby enabling the population of peripheral lymphoid organs and T cell-mediated colitis.
INTRODUCTION
Integrin-mediated lymphocyte adhesion plays an essential role in lymphocyte development and their capacity to populate lymphoid organs and sites of inflammation. Furthermore, by playing a critical accessory role in the formation of immunological synapses, integrins facilitate processes such as cytotoxic killing and antigen presentation. Integrins αLβ2 (LFA-1), α4β1 (VLA-4) and α4β7 (LPAM) are the lymphocyte integrins most commonly involved in these functions and each has served as a therapeutic target in autoimmune and inflammatory diseases(Hogg et al., 2011; Ley et al., 2016). Lymphocyte integrins are constitutively in a low-affinity state until agonist stimulation induces high-affinity, a process operationally defined as “integrin activation”(Luo et al., 2007; Takagi et al., 2002; Xiong et al., 2001). Integrin activation is essential to their capacity to mediate the lymphocyte functions enumerated above(Hynes, 2002; Ley et al., 2007).
Stimulation via agonists such as chemokines or cytokines initiates a spectrum of intracellular signaling pathways to trigger integrin activation. These pathways ultimately converge upon the binding of talin1 to the integrin β cytoplasmic tail(Kim et al., 2011; Lefort et al., 2012; Tadokoro et al., 2003; Ye et al., 2010), a final common step in integrin activation. The small GTPase Rap1 is a dominant hub in the signaling pathways that control the talin-integrin-interaction(Bos, 2005). In conventional T cells, Rap1 utilizes RIAM as an effector for integrin activation, whereas in CD4+Foxp3+ regulatory T cells, another MRL protein, Lamellipodin (LPD), makes a more important contribution(Sun et al., 2021). We previously showed that the MRL Protein-Integrin-Talin (MIT) complex forms the “sticky fingers” that drive cell protrusion and enable migratory pathfinding(Lagarrigue et al., 2015).
RIAM is abundant in hematopoietic cells. Unlike talin1 deletion, germline loss of RIAM in mice does not affect development, hemostasis, or platelet integrin function(Klapproth et al., 2015; Stritt et al., 2015; Su et al., 2015). However, RIAM plays an important role in the activation of β2 and β7 integrins in neutrophils, macrophages and T cells(Boussiotis et al., 1997; Klapproth et al., 2015; Medrano-Fernandez et al., 2013; Su et al., 2015; Sun et al., 2021). β1 and β3 integrin functions are less affected by the absence of RIAM in these leukocytes(Klapproth et al., 2015; Su et al., 2015). RIAM-deficient mice exhibit significant leukocytosis associated with leukocyte adhesion deficiency and impaired leukocyte extravasation. The apparently normal development and lack of bleeding in RIAM null mice, combined with their protection in models of autoimmune disease such as inflammatory bowel disease(Sun et al., 2021) or type I diabetes(Lagarrigue et al., 2017), suggests that targeting the regulation of the RIAM MIT complex could serve as a strategy to inhibit pathological inflammation or autoimmunity.
Here we analyzed the mechanisms that orchestrate the function of the MIT complex by using a tandem affinity purification tag to isolate the native complex and mass spectrometry to identify associated proteins. We focused our attention on Phostensin (PTSN), an actin-binding regulatory subunit of protein phosphatase 1 (PP1) encoded by the PPP1R18 gene(Kao et al., 2007) because PTSN is mainly expressed in leukocytes(Lin et al., 2011) and shares similar tissue distribution with RIAM. Here, we report that PTSN is physically associated with MIT complex and that it stabilizes the complex by mediating the dephosphorylation of Rap1 thereby preserving Rap1 activity and enabling integrin activation. We generated viable and fertile Ppp1r18-/- mice that exhibited impaired population of peripheral lymphoid tissues and defective activation of T cell integrins αLβ2 and α4β7. Because Ppp1r18-/- mice appear healthy yet have a defect in lymphocyte integrin function, we suggest that PTSN may be a therapeutic target in autoimmune and inflammatory diseases. In support of this idea, Ppp1r18-/- T cells manifested reduced capacity to induce colitis in an adoptive transfer model. In sum, we identify PTSN as a new regulator of lymphocyte integrin-mediated functions, show that it acts via stabilization of the RIAM MIT complex by dephosphorylating Rap1, and identify it as a potential therapeutic target in T cell-mediated diseases.
METHODS
Cell lines and plasmids
U2-OS, HEK293 and 293A cells were grown in DMEM supplemented with 10%(v/v) FBS, non-essential amino acids, 1x Penicillin/Streptomycin, and 2mM L-glutamine at 37°C in a 5% CO2 incubator. Jurkat T cells were from ATCC and cultured in RPMI supplemented with 10%(v/v) FBS, non-essential amino acids, 1x Penicillin/Streptomycin, and 2 mM L-glutamine. U2-OS stable cell lines expressing αΙΙb-streptavidin(SBP)-β3 or αΙΙb-SBPβ3(D119A) were generated by lentiviral infection of αIIb-SBP with β3 or αIIb-SBP with β3(D119A), respectively. Monoclonal cell populations were selected by flow cytometry.
Mammalian expression plasmids encoding full-length integrin αIIb-SBP and β3 (WT, wild-type) or (D119A) were cloned into pcDNA3.1(-). Myc-, Myc-mCherry- or DsRed-tagged human β- (aa 1-613) and α-PTSN (aa 249-613) were cloned into pcDNA3.1(-). Full-length RIAM cDNA was cloned into p3xFlag-CMV-7.1 (Sigma-Aldrich, St. Louis, MO). Constructs encoding EGFP-tagged full-length talin1 WT were previously described(Wegener et al., 2007). Coding sequences of β- and α- PTSN were cloned into pEGFP-C1 and p3xFlag-CMV-7.1 (Sigma-Aldrich). β- PTSN 4A mutation (residues K539, I540, S541 and F542 into four Ala) predicted to block binding to protein phosphatase 1 (PP1)(Kao et al., 2007) was produced by site-directed mutagenesis. Bicistronic constructs expressing Flag-RIAM together with either Myc-His-Rap1a WT or S180A mutant were generated into pcDNA3.1(-). Plasmid encoding EGFP-tagged PP1 catalytic subunit γ was obtained from Addgene (Addgene plasmid # 44225). Expression of PTSN was silenced in U2-OS and 293A cells by lentiviral transduction of a pLKO1 shRNA against both β- and α-PTSN (TRCN0000282572, Sigma-Aldrich). pLKO1 shRNA (SHC016V, Sigma-Aldrich) was used as a control.
Antibodies and reagents
Antibodies against talin (8d4, Sigma-Aldrich) and Flag (M2) were from Sigma-Aldrich. Antibody against DsRed (sc-33354) was from Santa Cruz Biotechnology (Dallas, TX). Anti-integrin αIIb (PMI-1), anti-β3 (Rb8257 and Rb8053)(Frelinger et al., 1990; Frelinger et al., 1988), activated anti-αIIbβ3 (Pac1)(Shattil et al., 1985) were described previously. Polyclonal anti-EGFP antibody was produced in rabbits immunized with recombinant EGFP. Anti-Flag M2 affinity gel was purchased from Sigma-Aldrich, and Pierce Streptavidin Plus UltraLink Resin and Pierce Monomeric Avidin Agarose were from Thermo Fisher Scientific (Rockford, IL). 3xFlag peptide (MDYKDHDGDYKDHDIDYKDDDDK) was synthesized by United BioSystems Inc (Hemdon, VA). Fluorophore-conjugated antibodies against CD3 (17A2, 2C11), B220 (RA3-6B2), CD49d (9F10), CD11a (M17/4), CD29 (HMβ1-1), CD18 (M18/2) and β7 (FIB 504) were purchased from BioLegend (San Diego, CA). Secondary AlexaFluor-labelled antibodies were from Jackson ImmunoResearch (West Grove, PA). MojoSort mouse CD4 T cell isolation kit and SDF-1α were from BioLegend. Recombinant human VCAM-1-Fc, mouse ICAM-1-Fc and VCAM-1-Fc were from BioLegend. Recombinant mouse MAdCAM-1-Fc was from R&D Systems (Minneapolis, MN).
Antibody against β-PTSN was from Santa Cruz Biotechnology. To generate antibody against α-PTSN, mouse PTSN C-terminus (a.a. 429-594) was cloned into pETM-11 vector. His-PTSN fusion protein was solubilized from inclusion bodies with 6M urea and purified under denaturing conditions using His-Bind resin. The protein was dialyzed against PBS multiple times and used to raise polyclonal rabbit antisera (Abgent, San Diego, CA).
Purification of MIT complex by split-tandem affinity purification
Sub-confluent U2-OS cells stably expressing integrin αIIb-SBP and β3(D119A) were transfected with plasmids encoding Flag-RIAM or encoding the indicated Flag-tagged bait for 24-48 hrs. After washing with PBS, cells were harvested and lysates were prepared in lysis buffer (50 mM Tris-HCl (pH7.4), 100 mM NaCl, 0.5% NP-40, 0.5 mM MgCl2, 0.5 mM CaCl2, 0.2 mM GMP-PNP, 10 mM N-Ethylmaleimide, 1 μM Calpeptin, PhosStop and protease inhibitor cocktails). Cell lysates were centrifuged at 22,000 g for 30 min. The supernatant was collected and precleared with anti-mouse IgG coupled to agarose beads (Sigma-Aldrich) for 2 hrs at 4°C. Cell lysates were then loaded into anti-Flag affinity column and incubated for 4 hrs at 4°C. Bound proteins were eluted with 200 μg/mL 3xFlag peptide after washing with lysis buffer. The eluate was subsequently loaded into monomeric avidin agarose or streptavidin plus ultralink column (Thermo Fisher Scientific) and incubated for 2 hrs at 4°C. The associated proteins were washed and eluted by 5 mM D-biotin or boiled in Laemmli buffer for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting.
Identification of MIT-associated proteins
The gel piece was transferred to a siliconized tube and washed and unstained in 200 µL 50% methanol overnight. The gel pieces were dehydrated in acetonitrile, rehydrated in 30 µL of 10 mM dithiothreitol in 0.1 M ammonium bicarbonate and reduced at room temperature for 30 min. The DTT solution was removed and the sample alkylated in 30 µL of 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room temperature for 30 min. The reagent was removed, and the gel pieces dehydrated in 100 µL acetonitrile. The acetonitrile was removed, and the gel pieces rehydrated in 100 µL of 0.1 M ammonium bicarbonateand the pieces completely dried by vacuum centrifugation. The gel pieces were rehydrated in 20 ng/µL trypsin in 50mM ammonium bicarbonate and digested overnight at 37 °C and the peptides formed extracted from the polyacrylamide in two 30 µL aliquots of 50% acetonitrile/5% formic acid. These extracts were combined and evaporated to 15 µL for MS analysis performed as previously described(Goldfinger et al., 2007).
Immunoprecipitation
Cells were harvested and lysed by NP-40 lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% NP-40, 0.5 mM CaCl2, 0.5 mM MgCl2, 1 μM calpeptin, protease inhibitor cocktail) on ice for 15 min. The lysate was centrifuged at 14,000 rpm at 4°C for 15 min. Protein concentration of the lysate was determined by using a BCA assay (Thermo Fisher Scientific). The lysate was immunoprecipitated using designated primary antibodies with protein G resin (GenScript, Piscataway, NJ), or anti-Flag M2 affinity agarose gel at 4°C. Then, immune complexes were washed for extended times with the lysis buffer and separated by SDS-PAGE gels. Proteins on the gel were transferred to nitrocellulose membrane and probed with indicated primary and secondary antibodies.
Flow cytometry
Cells isolated from mouse tissues were washed and resuspended in PBS containing 0.1% BSA and stained with conjugated antibody for 30 min at 4 °C. Then cells were washed twice before flow cytometry analysis using an Accuri C6 Plus (BD Biosciences, Franklin Lakes, NJ). Data were analysed using FlowJo software (BD Biosciences).
Integrin activation assay
The activation state of αIIb(R995A)β3 was assessed by measuring the binding of the ligand mimetic anti-αIIbβ3 monoclonal antibody PAC1 in two-color flow cytometric assays as described previously(O’Toole et al., 1994). PTSN expression in HEK293 stable cells expressing integrin αIIb(R995A)β3 was silenced by shRNA and the cells were transfected with shRNA-resistant EGFP-PTSN or EGFP vector as a transfection marker. After 24 hrs, cells were suspended and stained with PAC1. Then, cells were washed and analyzed by flow cytometry. Background binding was measured in presence of the αIIbβ3 antagonist integrilin (10 μM) whereas maximum binding was quantified upon addition of the anti-LIBS6 monoclonal antibody(Frelinger et al., 1991) against αIIbβ3 (2. μM). To measure the activation of α4β1(Rose et al., 2000), cells (5×105) were resuspended in HBSS buffer (Mediatech, Manassas, VA) containing 5 μg/mL human VCAM-1-Fc for 30 min at 37°C. Afterward, cells were washed with 0.5 mL of HBSS buffer, then resuspended in the same buffer containing AlexaFluor647-conjugated anti-human antibody and incubated for 30 min on ice. Cells were washed twice prior to analysis by flow cytometry. Background binding was measured in presence of 5 mM EDTA whereas maximum binding was quantified upon addition of 1mM MnCl2.
Flow chamber assay
A polystyrene Petri dish was coated with 10 μL of MAdCAM-1/Fc, VCAM-1/Fc or ICAM-1/Fc alone or with SDF-1α (2 μg/ml) in coating buffer (PBS, 10 mM NaHCO3, pH9.0) for 1 hr at 37°C followed by blocking with 2% BSA in coating buffer for 1 hr at 37°C. Cells were diluted 106 cells/mL in HBSS (10 mM HEPES, 1 mM Ca2+/Mg2+) and immediately perfused through the flow chamber at a constant flow rate of 2 dyn/cm2. Adhesive interactions between the flowing cells and the coated substrates were assessed by manually tracking the motions of individual cells for 1 min as previously described(Sun et al., 2020; Sun et al., 2014). The motion of each adherent cell was monitored for 10 sec following the initial adhesion point, and two categories of cell adhesion were defined: rolling adhesion for cells with rolling motions for >10 sec with a velocity >1 μm/sec, whereas cells that remained adherent and stationary for >10 sec with a velocity <1 μm/sec were defined as arrested adherent cells. The total adherent cell number includes both rolling adherent and firm adherent cells.
Mice
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, San Diego, and were conducted in accordance with federal regulations as well as institutional guidelines and regulations on animal studies. All mice were housed in specific pathogen-free conditions. C57BL/6J (CD45.1), C57BL/6J (CD45.2), and Rag1-/- mice were from The Jackson Laboratory (Bar Harbor, ME). For experiments, 8-12-week-old mice were used. All injections of cells were performed during the light cycle. All experiments were performed by comparing mutant mice with littermate controls. Mononuclear cells were isolated from mesenteric lymph node (MLN), Peyer’s patch (PP), peripheral lymph node (PLN), spleen (SP) and colonic lamina propria as previously described. Ppp1r18-/- mice were generated using a CRISPR/Cas9 approach at the University of California Irvine Transgenic Mouse Facility. The complex Cas9 mRNA/sgRNA/tracRNA (3μM) was injected into the pronuclei of C57BL/6N embryos. Surviving embryos were implanted into ICR pseudo-pregnant females and pregnancies went to full term. Tissue biopsies for genomic DNA were taken from pups between 7-10 days. Mice were genotyped by PCR using forward primers 5’-ggacgacctgggacatagataca-3’ and reverse primers 5’- ttttcacacgccttcacaggta-3’ followed by Sanger sequencing using forward primers 5’- tccagacagcaggaagaggaag-3’ and reverse primers 5’- tctacccagtcaggcatggt-3’.
Founders were selected and backcrossed to the C57BL/6J strain (>5 times) to establish the Ppp1r18-/- strain. To generate the Ppp1r18β-/- mice, a similar CRISPR/Cas9 approach was used to inject a complexed Cas9 mRNA/sgRNA (3 μM) into the pronuclei of C57BL/6NHsd embryos at the University of California San Diego Transgenic Mouse Facility. One homozygous pup harbouring a 10 base pair deletion resulting in the appearance of a downstream premature stop codon was selected and backcrossed to the C57BL/6J strain (>5 times) to establish the Ppp1r18β-/- strain, which only lacks the β form of PTSN. Mice were genotyped by PCR using forward primers 5’-ctgagcagagacccactgaaag-3’ and reverse primers 5’- ggatctggcttctgagtttgtgta-3’ and amplicons were ran in 6% TBE gels (Life Technologies, Carlsbad, CA).
Mouse colitis models
The adoptive T cell transfer model was described before(Sun et al., 2020). Briefly, 8-10-week-old mice were used. 5×105 CD4+CD25-CD45RBhigh conventional T cells from WT or Ppp1r18-/- mice were injected intraperitoneally into Rag1-/- mice(Mombaerts et al., 1992). All comparisons were made between littermates. Mouse body weight was measured every day and values are shown as a percentage of the original weight. During the duration of the experiment, we assessed the clinical progression of colitis by daily blinded scoring a disease activity index (DAI) by 2 independent investigators. The DAI is the combined score of body weight loss, stool consistency, and rectal bleeding and prolapse as follow: 1) Weight loss: 0 (no loss), 1 (1-5%), 2 (5-10%), 3 (10-20%), 4 (>20%); 2) Stool consistency: 0 (normal),1 (soft), 2 (very soft), 3 (diarrhea); 3) Rectal bleeding: 0 (none), 1 (red), 2 (dark red), 3 (gross bleeding); 4) Rectal prolapse: 0 (none), 1 (signs of prolapse), 2 (clear prolapse), 3 (extensive prolapse). Mice were sacrificed at week 15.
Histology
Formalin-fixed, paraffin-embedded Swiss-rolled colon sections of 4-mm thickness were mounted on glass slides and followed by hematoxylin and eosin staining or periodic acid–schiff staining. Images were acquired with a Nanozoomer Slide Scanner (Hamamatsu, Hamamatsu City, Japan). Blinded histological scoring was performed by 2 investigators based on the method described previously(Erben et al., 2014) and total scoring range is 0-12.
Blood counts
Peripheral blood was collected from the retro-orbital plexus and transferred to tubes containing K+.EDTA. Cell counts were performed using a Hemavet 950FS Hematology System programmed with mouse-specific settings (Drew Scientific. Miami Lakes, FL). All samples were tested in duplicate, and the mean for each animal was plotted.
Real-time quantitative PCR analyses
Total RNA was isolated from colon using tissue homogenizer (JXFSTPRP-24, ThunderSci, Shanghai, China) and TRIzol reagent according to the manufacturer’s protocol (Thermo Fisher Scientific). For gene expression analysis, single-stranded cDNA was produced from 10 μg total RNA of colon using SuperScript III First-Strand synthesis and oligo-dT primers according to the manufacturer’s protocol (Thermo Fisher Scientific). Kappa SybrFast qPCR kit (Kapa Biosystems, Wilmington, MA) and thermal cycler (CFX96 Real-Time System; Bio-Rad, Hercules, CA) were used to determine the relative levels of the genes analysed (primer sequences are shown in Table 1) according to the manufacturer’s protocol. The 2-ΔΔCT method was used for analysis, and data were normalized to GAPDH. Control values (Rag1-/- mice injected with PBS) were set to 1 for comparisons.
Statistics
Statistical significance was assayed by a two-tailed t-test for single comparisons. ANOVA with a Tukey post hoc test was used to assay statistical significance for multiple comparisons. A p value <0.05 was considered significant.
Supplementary Material: Supplementary Table 1 contains the proteomic analysis of the purified MIT complex, Supplementary Figures 1-3.
RESULTS
PTSN is a component of the MIT complex
To isolate an MIT complex, we devised a split tandem affinity purification method that isolates the native complex formed prior to ligand engagement (Fig. 1A). We transfected Flag-RIAM into U2-OS cells stably expressing integrin αIIb-streptavidin-binding peptide (SBP) and β3(D119A) to form ligand binding-defective αIIb(SBP)β3(D119A). RIAM and associated proteins were isolated by anti-Flag affinity chromatography of cell lysates and the native proteins were eluted with 200 μM Flag peptide. The eluate was then passed through a streptavidin column to bind the αIIb(SBP) and the native MIT complex was eluted with 5 mM biotin. In five experiments an average of 100 µg of complex was isolated from 2×107 cells. The eluate of the streptavidin column was fractionated on SDS-PAGE and major protein components were visualized by immunoblotting with antibodies against talin, integrin, or RIAM (right, Fig. 1B). In a proteomic analysis (Supplementary Table 1), we noted that PTSN was a prominent component of the isolated complex. PTSN, encoded by PPP1R18, is an actin binding regulatory subunit of protein phosphatase 1 (PP1). PTSN shares these biochemical functions with Phactr4, which modulates integrin signaling and cofilin activity to coordinate enteric neural crest cell migration(Zhang et al., 2012). PTSN was also present in the LPD MIT complex (Fig. 1C). Importantly, PTSN was absent from the eluates of both Flag and streptavidin columns in the absence of Flag-RIAM (Fig. 1D).
Both isoforms of PTSN regulate integrin activation
To examine the role of PTSN in integrin function, we used 293 cells constitutively expressing a recombinant talin-dependent(Tadokoro et al., 2003) activated integrin αIIb(R995A)β3. Silencing PTSN inhibited activation and co-expressing shRNA-resistant PTSN rescued activation of this integrin (Fig. 2A,B). PTSN has a short and long isoform, termed α and β respectively(Lin et al., 2014), and both contain actin and PP1 binding domains (Fig. 2C). Either isoform associated with RIAM (Fig. 2D) and could rescue integrin activation (Fig.2E).
Analysis using the human proteome map (http://www.humanproteomemap.org/batch.php) confirmed that both RIAM and PTSN are particularly well expressed in lymphoid cells (Fig. 3A). We therefore used CRISPR/Cas9 mutagenesis to inactivate PPP1R18 in the Jurkat T cell line and observed a marked reduction in basal and phorbol myristate acetate (PMA)- stimulated activation of an endogenous integrin (Fig. 3B,C). In both cases activation was rescued by re-expressing either α- or β-PTSN. Thus, both isoforms of PTSN regulate integrin activation in a model system and a human T cell line.
PTSN regulates the assembly of the MIT complex
To investigate how PTSN regulates integrin activation, we tested the role of PTSN in the assembly of the RIAM MIT complex. We silenced PTSN expression in cells expressing recombinant Flag-RIAM and integrin αIIbβ3 and purified Flag-RIAM by immuno-affinity chromatography (Fig. 4A). Silencing PTSN dramatically reduced the association of both integrin β3 and talin with RIAM, indicating disassembly of the MIT complex. The complex was assembled when shRNA-resistant β-PTSN was co-expressed with the PTSN shRNA (Fig. 4 B-E). Thus, PTSN regulates the assembly of the MIT complex.
Activated Rap1 drives the formation of the MIT complex and results integrin activation, leading us to examine the potential role of PTSN in regulating Rap1. Protein kinase A (PKA) phosphorylation of Rap1 inhibits Rap1 activity by decreasing GTP loading and disrupting membrane localization(Takahashi et al., 2013). To assess whether silencing PTSN could affect Rap1 phosphorylation, Rap1 was immunoprecipitated from cells in which endogenous PTSN expression was silenced by PTSN-specific shRNA. Rap1 phosphorylation was assessed by an antibody specific for phosphorylated PKA substrates. Silencing endogenous PTSN led to increased Rap1 phosphorylation and overexpression of shRNA resistant PTSN abolished Rap1 phosphorylation (Fig. 5A). To examine the specificity of increased phosphorylation by PTSN silencing, we examined cAMP Response Element-Binding Protein (CREB), an abundant PKA substrate. Silencing PTSN expression had no effect on Forskolin-induced CREB phosphorylation (Fig. 5B). Rap1a is phosphorylated at COOH-terminal Serine residue 180 by PKA both in vivo and in vitro(Takahashi et al., 2013). To directly address if Rap1 de-phosphorylation is important for RIAM MIT complex assembly, we asked if phosphorylation resistant Rap1a mutant (S180A) could support RIAM MIT assembly when PTSN was silenced. We generated IRES (internal ribosome entry site)-based bicistronic construct (Fig. 5C) expressing both Flag-RIAM and Myc-Rap1a(S180A) and transfected it into the cells expressing integrin αIIbβ3 and Flag-RIAM. Expression of Rap1a(S180A) but not wild type Rap1a restored the association of integrin β3 with RIAM, indicative of the intact RIAM MIT complex, in PTSN silenced cells (Fig. 5C). Importantly, silencing PTSN did not affect the similar expression levels of Rap1a(S180A) or of Rap1a, nor did it affect expression of Flag-RIAM or α-tubulin (Fig. 5C). Together, these data show that PTSN stabilizes the MIT complex and thus, integrin activation by antagonizing the phosphorylation of Rap1.
PTSN is a PP1 regulatory subunit and we confirmed that mutation of the PP1 catalytic subunit (PP1C) binding site disrupted its association with PTSN (Fig. 5E). This PP1C binding-defective mutant PTSN was unable to support integrin activation (Fig. 5F,G). PTSN is an actin associated protein(Lai et al., 2009; Wang et al., 2012), therefore we tested if silencing PTSN expression could affect the phosphorylation of regulators of actin dynamics whose activities are controlled by phosphorylation. Silencing of PTSN increased the phosphorylation of Cofilin (Supplementary Fig. 1A) and VASP (Supplementary Fig. 1B). Overexpression of shRNA resistant PTSN, either α or β isoforms, abolished the increased phosphorylation of cofilin (Supplementary Fig. 1C). Thus, in addition to preserving the activity of Rap1 and thus integrins, PTSN can induce de-phosphorylation of regulators of actin dynamics.
Ppp1r18-/- mice are viable, fertile, and manifest reduced integrin activation
The foregoing data show that PTSN, a component of the MIT complex, stabilizes the assembly of that complex and resulting integrin activation by regulating the phosphorylation of Rap1. We therefore created Ppp1r18-/- mice to assess a potential role for PTSN in vivo. We devised three different CRISPR/Cas9 strategies to inactivate Ppp1r18. We first used a combination of two different gRNAs to introduce 10 stop codons downstream of the codon for the initiator Methionine residue 428 of the α form of PTSN (Fig. 6A). To eliminate possible off target effects, we used different guide RNAs to delete the whole coding sequence of Exon 1 that contains initiator Methionine of both α and β forms of PTSN (Supplementary Fig. 2A). In both cases, homozygous Ppp1r18-/- offspring mice were viable and fertile and their splenocytes lacked both PTSN α and β isoforms (Fig. 6B, Supplementary Fig. 2B), and exhibited no change in expression of integrins αL, α4, β2, β1, or β7 (Fig. 6C) or of RIAM (Fig. 6D) in comparison with wild type littermates. To specifically inactivate the β form of PTSN, we used a gRNA upstream of the alternative translation start site at Methionine residue 428 resulting in a 10-base pair deletion which provokes a premature stop codon (Supplementary Fig. 3A) and absence of the β-PTSN form, but intact expression of the α form, in splenocytes (Supplementary Fig. 3B). Homozygotes of Ppp1r18β-/- mice, lacking only the β form of PTSN, were viable and fertile and their splenocytes expressed a normal complement of integrins αL, α4, β2, β1, or β7 (Supplementary Fig. 3C) or of RIAM (Supplementary Fig. 3D).
Ppp1r18-/- mice of both genotypes lacking both forms of PTSN, exhibited a ∼two-fold increase in blood leukocytes that affected both lymphocytes and neutrophils (Fig. 6E) and an accompanying decrease in T and B cells in peripheral lymphatic organs (Fig. 6F). The leukocytosis in these mice combined with a reduction in lymphocytes in peripheral lymphatic organs is similar to that observed in RIAM deficient Apbb1ip-/- mice(Klapproth et al., 2015) (Su et al., 2015) and is explained by the reduction in T cell (Fig. 6G-I) and B cell (Fig. 6J) integrin activation. Furthermore, Ppp1r18-/- splenocytes exhibited increased phosphorylation of cofilin (Supplementary Fig. 1D) and VASP (Supplementary Fig. 1E) similar to that observed in U2-OS cells (Supplementary Fig. 1A,B). We were unable to immunoprecipitate sufficient Rap1 from splenocytes to analyze its phosphorylation. Thus, PTSN enables activation of lymphocyte integrins and population of peripheral lymphoid organs.
In sharp contrast to Ppp1r18-/- mice, Ppp1r18β-/- mice exhibited blood counts indistinguishable from those of wild type littermates (Supplementary Fig. 3E). Consistent with the absence of leukocytosis, Ppp1r18β-/- T cells exhibited similar activation of T cell αLβ2 and α4β1 integrins as judged by adhesion on ICAM-1, or VCAM-1 respectively (Supplementary Fig. 3G,H). The preservation of lymphocyte integrin function was manifested phenotypically by the normal population of peripheral lymphatic tissues by B cells and T cells (Supplementary Fig. 3F). Because Ppp1r18β-/- mice exhibited no obvious phenotype, α-PTSN is sufficient for these lymphocyte functions.
Ppp1r18-/- T cells exhibit reduced capacity to induce colitis
Because PTSN null mice were apparently healthy, yet exhibited defects in T cell integrin activation, we hypothesized that their T cells might lack the capacity to provoke auto-immune inflammation. We induced experimental autoimmune colitis(Sun et al., 2020) by infusing CD4+CD25-CD45RBhigh T cells (TCONV) from Ppp1r18-/- mice or WT mice into Rag1-/- recipient mice. Rag1-/- mice injected with WT TCONV manifested a progressive loss in bodyweight 20-30 days after the infusion (Fig. 7A). Half of these mice died by 100 days (Fig. 7B). In contrast, Rag1-/- mice injected with Ppp1r18-/- TCONV exhibited a little loss of bodyweight and only 10 % of these mice died by 100 days (Fig. 7A,B).
Histologically, wild-type TCONV infusion led to a severe colitis in Rag1-/- mice, with almost complete loss of crypts, dense infiltrates of leukocytes in both mucosa and submucosa, and thickening of the bowel wall (Fig. 7C). By contrast, the inflammatory infiltrates in the Rag1-/- mice infused with Ppp1r18-/- TCONV were much reduced and less tissue damage was observed (Fig. 7C). Blinded histological scoring for inflammatory cell infiltrates and epithelial damage confirmed a reduction in the severity of colitis in Ppp1r18-/- TCONV infused Rag1-/- mice (Fig. 7D). The difference in inflammatory cell infiltration between Ppp1r18-/- and wild-type TCONV infused Rag1-/- mice was confirmed by the reduction in colonic expression of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6, IFN-γ and IL-17A) in Ppp1r18-/- TCONV recipients (Fig. 7E). Thus, the defect in integrin activation in PTSN null CD4+ T cells is associated with impaired capacity to induce intestinal inflammation.
DISCUSSION
The MIT complex plays an essential role in leukocyte integrin activation and thus in the trafficking and functions of lymphocytes in immunity and immunopathology(Su et al., 2015). Here we report that an actin binding regulatory subunit of PP1, PTSN, encoded by the Ppp1r18 gene(Kao et al., 2007), stabilizes the MIT complex by mediating the dephosphorylation of Rap1 thereby preserving Rap1 activity. The stabilization of the MIT complex enables lymphocyte integrin activation and cell adhesion. Ppp1r18-/- mice are viable, fertile, and apparently healthy. These mice exhibit reduced lymphocyte population of peripheral lymphoid tissues associated with lymphocytosis in peripheral blood, findings ascribable to defective activation of lymphocyte integrins αLβ2 and α4β7. Furthermore, Ppp1r18-/- T cells manifested reduced capacity to induce colitis in an adoptive transfer model. Thus, PTSN is a regulator of T cell integrin-mediated functions that stabilizes the RIAM MIT complex by dephosphorylating Rap1. In spite of this defect in T cell integrin function Ppp1r18-/- mice appear healthy suggesting that PTSN may be a therapeutic target in T cell mediated autoimmune diseases.
PTSN is physically associated with MIT complex and enables integrin activation by mediating de-phosphorylation of Rap1, thereby stabilizing the complex. PTSN was associated with the MIT complex formed by a ligand binding-defective integrin(Loftus et al., 1990) explaining why this association can form in non-adherent lymphocytes. Activation of integrins in lymphocytes requires Rap1(Su et al., 2015) and we find that PTSN helps maintain Rap1 in a functional non-phosphorylated state. In particular, we find that silencing of phostensin destabilizes the MIT complex and this destabilization can be prevented by a non-phosporylatable mutant of Rap1a. In addition to blocking membrane association of Rap1, Rap1 phosphorylation results in prolonged activation of ERK 1/2(Takahashi et al., 2017), an event that can also suppress integrin activation(Hughes et al., 1997) and assembly of the MIT complex(Lagarrigue et al., 2015). Thus, PTSN joins Phactr4(Zhang et al., 2012) as actin-associated protein phosphatase 1 regulators that modulate integrin functions.
Ppp1r18-/- mice exhibited lymphocytosis accompanied by a reduced population of peripheral lymphoid organs, a phenotype indicative of a failure of lymphocyte migration out of the blood as a consequence of reduced integrin activation. Cell migration requires anterior-posterior polarization of processes such as integrin activation and of actin dynamics to support directional migration(Ridley et al., 2003). The MIT complex is localized to and mediates the protrusions that form at the leading edge of migrating cells(Lagarrigue et al., 2015; Lee et al., 2013), a site at which α4 integrins can anchor Type I Protein Kinase A (PKA)(Lim et al., 2007; Lim et al., 2008) to enable PKA activation. PKA phosphorylation of RhoA forms protrusion-retraction pacemaker at the leading edge of migrating cells(Tkachenko et al., 2011). PTSN, by opposing PKA-mediated Rap1 phosphorylation, can preserve stability of the MIT complex at sites of increased PKA activity such as the leading edge(Lim et al., 2008). Conversely, over-expression of phospho-resistant Rap1 can arrest migration(Takahashi et al., 2013), suggesting that the dynamic interplay between PKA and PTSN in Rap1 phosphorylation can contribute to the protrusion-retraction cycles that govern cell migration.
Pre-clinical research identified inhibition of ligand binding to lymphocyte integrins as a potential therapeutic target in autoimmune and inflammatory diseases(Dustin, 2019; von Andrian and Engelhardt, 2003). This idea was validated by the success of vedolizumab anti-α4β7 in IBD(Feagan et al., 2013), natalizumab anti-α4β1 in multiple sclerosis(Rice et al., 2005), and efalizumab anti-αLβ2 in psoriasis(Dedrick et al., 2002). Serious mechanisms-based toxicities such as progressive multifocal leukoencephalopathy have limited the use of natalizumab and efalizumab. Inhibition of integrin signaling can preserve some integrin function and can therefore ameliorate mechanism-based toxicities of complete blockade of integrin function(Feral et al., 2006; Petrich et al., 2007). Talin and Rap1 are major elements in signaling pathways that activate leukocyte integrins; however, global loss of talin-1 or combined loss of Rap1a and Rap1b, leads to embryonic lethality in mice(Calderwood et al., 2013; Li et al., 2007). RIAM plays a key role in Rap1- dependent talin-mediated activation of αLβ2 and α4β7 integrins(Su et al., 2015; Sun et al., 2021) in most leukocytes and lack of RIAM leads to no obvious developmental defects or abnormalities in platelet functions(Klapproth et al., 2015; Stritt et al., 2015; Su et al., 2015). Here we show that PTSN supports Rap1- induced assembly of the RIAM-talin-integrin complex required for integrin activation. In the absence of PTSN a defect in lymphocyte trafficking is associated with the inability of T cells to provoke experimental colitis. Importantly, like RIAM, PTSN is expressed at low levels in platelets (Fig. 3A) and we observed no gross bleeding tendency or anemia in Apbb1ip-/- mice. Thus, PTSN inhibition may provide a means to blunt immune-mediated inflammation with less mechanism-based toxicity than direct blockade of ligand binding to leukocyte integrins.
Conflict-of-interest disclosure
The authors declare no competing financial interests.
Author Contributions
M.H.G conceived the study, designed experiments, analyzed data, and wrote the manuscript. H.-S.L., H.S. and F.L. designed and executed experiments, analyzed data, and wrote the manuscript. J.W.F and N.E.S. performed mass spectroscopic analysis. A.R.G designed and produced antigen, and tested the antibody produced. M.H.G, H.-S.L., H.S., F.L., and A.R.G edited the manuscript.
SUPPLEMENTARY FIGURE LEGENDS
Acknowledgements
We thank the UCI and the UCSD Transgenic Mouse Facilities for design help and production of CRISPR modified mice; Jennifer Santini for microscopy technical assistance; and the UCSD School of Medicine Microscopy Core (NINDS P30NS047101). The UCI TMF is a shared resource funded in part by the Chao Family Comprehensive Cancer Center Support Grant (P30CA062203) from the National Cancer Institute. This work was supported by grants HL 139947 and HL 151433 to MHG and by the American Heart Association Career Development Award 18CDA34110228 (F.L.) and Scientist Development Grant 14SDG18440023 (HSL).
Footnotes
Updated order of authors online