Control of cell adhesion and migration by podocalyxin. Implication of Rac1 and Cdc42.

Podocalyxin (PODXL) is a type I membrane sialomucin, originally described in the epithelial cells (podocytes) of kidney glomeruli. PODXL is also found in extra-renal tissues and in certain aggressive tumors, but its precise pathophysiological role is unknown. Expression of PODXL in CHO cells enhances their adhesive, migratory and cell-cell interactive properties in a selectin and integrin-dependent manner. We aimed at defining the PODXL domains responsible for those cell responses. For this purpose we have analyzed the cell adhesion/migration responses to deletion mutants of human PODXL, and the correlation with the activities of Rac1 and Cdc42 GTPases. The results obtained indicate that integrity of the PODXL ectodomain is essential for enhancing cell adhesion but not migration, while the integrity of the cytoplasmic domain is required for both adhesion and migration. Deletion of the carboxy-terminal DTHL domain (PODXL-ΔDTHL) limited only cell adhesion. The activities of Rac1 and Cdc42 GTPases parallel the PODXL-induced variations in cell adhesion and migration. Moreover, silencing the rac1 gene virtually abolished the effect of PODXL in enhancing cell adhesion.

Release of podocalyxin into the extracellular space. Role of metalloproteinases.

Podocalyxin (PODXL) is a type I membrane mucoprotein abundantly presented in the epithelial cells (podocytes) of kidney glomeruli where it plays an important role in maintaining the plasma filtration. PODXL is also expressed in other types of cells but its function is ignored. A recombinant soluble fragment of the PODXL ectodomain modifies the signaling of the membrane bound PODXL. Based on this antecedent, we aimed at investigating whether PODXL could be cleaved and released into the extracellular space as a soluble peptide. In this study, we used a fusion protein of human PODXL and green fluorescent protein expressed in CHO cells (CHO-PODXL-GFP) and a human tumor cell (Tera-1) inherently expressing PODXL. PODXL was detected by wide-field microscopy in the Golgi, the plasma membrane and in a vesicular form preferentially located at the leading edges of the cell and also progressing along the filopodium. We detected PODXL in the insoluble and soluble fractions of the extracellular medium of CHO-PODXL-GFP cells. Stimulation of protein kinase C (PKC) by Phorbol-12-myristate-13-acetate (PMA) enhanced the release of PODXL to the extracellular space whereas this effect was prevented either by inhibitors of PKC or specific inhibitors of matrix metalloproteinases. It is concluded that intact PODXL is released to the extracellular space as a cargo of microvesicles and also as a soluble cleaved fragment of ectodomain.

Ventricular enlargement associated with the panneural ablation of the podocalyxin gene.

Podocalyxin (Podxl) is a type I membrane mucin-protein of the CD34 family abundantly expressed in kidney epithelial cells (podocytes) where it plays a crucial functional role. Podxl is also expressed in tissues other than kidney, like in brain, but its function is ignored. To investigate the functional role of podocalyxin (Podxl) in brain we produced the specific brain-ablation of the Podxl gen in mice by crossing Podxl(floxed/floxed) mice, generated in our laboratory, to mice with pan-neural expression of recombinase Cre (Cre3). Podxl(-/-) mice show no apparent behavioral phenotype but their brains showed enlargement of ventricular volumes detected in vivo by MR imaging. The pattern of brain vasculature was of normal appearance but the thickness of the main carotid artery was significantly increased. Moreover, the histological analysis showed increased number of choroidal capillaries lining the ventricular spaces. These findings are analyzed in the light of the role likely played by podocalyxin in cell migration and cell-cell recognition during brain development and also on the consistent findings of increased ventricular spaces in human pathological disorders like schizophrenia.

Expression of podocalyxin enhances the adherence, migration, and intercellular communication of cells.

Podocalyxin (PODXL) is an anti-adhesive glycoprotein expressed abundantly in the epithelial cells of kidney glomeruli. In contrast, we report herein that expression of podocalyxin(GFP) (PODXL(GFP)) in CHO cells increased the adherence to immobilized fibronectin, spreading, and migration. The transient knockdown of PODXL or the expression of PODXL lacking the cytosolic carboxyterminal domain (PODXL-Delta(451)) inhibited cell adherence. Moreover, the effect of PODXL was prevented by the ectodomain of podocalyxin (PODXL-Delta(429)), by RGD peptides, or by inhibitors of the vitronectin receptor (alphavbeta3). CHO-PODXL(GFP) also showed adherence to human vascular endothelial cells (HUVEC), exhibiting polarization of granular PODXL and emission of long and thin, spike-like, protrusions with PODXL granules progressing along. We found PODXL colocalized with beta1 integrins at membrane ruffle regions on the leading edge of the cell and a blocking beta1 mAb prevented the spreading of cells. PODXL was also associated with submembrane actin in lamellipodia ruffles, or with vinculin at cell protrusions. The proadhesive effects of PODXL were absent in sialic acid deficient O-glycomutant CHO cells. To conclude, we present evidence indicating that human PODXL enhances the adherence of cells to immobilized ligands and to vascular endothelial cells through a mechanism(s) dependent on the activity of integrins.

Variations in platelet protein associated with arterial thrombosis.

INTRODUCTION: Hyperactivity of platelets has been associated with thrombotic episodes by molecular mechanisms not yet elucidated. The present work aimed at identifying whether the platelet protein content from patients who had suffered an arterial thrombosis episode differed from that of platelets obtained from normal healthy donors. METHODS: Differential platelet protein profiles were determined by 2-dimensional (2-D) gel electrophoresis and Western blot analysis of total platelet lysates. Identification of differentially expressed proteins was carried out by mass spectrometry (MALDI-TOF). RESULTS: We found a decreased platelet content of three protein spots in patients of arterial thrombosis: integrin linked kinase (ILK), fructose bisphosphate aldolase (aldolase) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) whereas the content of four other protein spots was increased: actin binding protein, coronine like (p57), non-muscle myosin heavy chain (NMMHC-A), pyruvate kinase M2 isoenzyme (PK) and phosphoglycerate kinase (PGK). The variations in ILK, GAPDH and PK were validated by Western blot analysis. The proteins showing a decreased platelet content in arterial thrombosis patients are associated with the cytoskeletal insoluble fraction and the detected increase in some proteins seems to be due to the generation of peptides caused by a limited proteolysis. Differences in the protein profiles of circulating platelets from arterial thrombosis were maintained months after the acute thrombotic event and disappear in the long term. CONCLUSIONS: The observed variations in some platelet proteins suggest the existence of a perturbation in the cytoskeletal organization and increased proteolysis, both indicative of a platelet pro-active state, persistent after the thrombotic event.

Role of transcription factor Sp1 and CpG methylation on the regulation of the human podocalyxin gene promoter.

BACKGROUND: Podocalyxin (podxl) is a heavily glycosylated transmembrane protein mainly found on the apical membrane of rat podocytes and also in endothelial, hematopoietic, and tumor cells. Despite of its interest no much is known about the transcriptional regulation of podxl in different cells. Thus, we aimed at studying the functional features of the 5'-regulatory region of the human Podxl gene. RESULTS: The promoter region of the human Podxl gene has been cloned and its structure and function were analyzed. The primary DNA sequence is rich in G+C and is devoid of TATA or CAAT boxes. The sequence contains recognition sites for several putative transcription factors; however, the basic promoter activity seems to rely entirely on Sp1 transcription factor since supershift analysis was positive only for this factor. The region encompassed by 66 to -111 nts conferred the minimal transcriptional activity that increases as the number of Sp1 sites augmented with the length of the promoter fragment. In Sp1-lacking insect cells the Podxl promoter constructs showed activity only if cotransfected with an Sp1 expression plasmid. Finally, mutation of the Sp1 sites reduced the promoter activity. We analyzed whether methylation of the CpG dinucleotides present in the first approximately 600 nts of the promoter region of Podxl could explain the variable rates of expression in different types of cells. Inactivation of methyltransferases by 5'-aza-2'deoxicitidine showed a dose-dependent increase in the podxl content. Moreover, in vitro methylation of the promoter constructs -111,-181 and -210 led to an almost complete reduction of the promoter activity. A correlation was found between the degree of methylation of the CpG promoter dinucleotides and the rate of podxl expression in different cell lines. CONCLUSION: Our results indicate that transcriptional regulation of Podxl is supported primarily by Sp1 site(s) and that DNA-methylation of the CpG promoter islands contributes to control the tissue specific expression of podxl.

alpha-Adrenergic-mediated activation of human reconstituted fibrinogen receptor (integrin alphaIIbbeta3) in Chinese hamster ovary cells.

This work reports the functional studies of CHO cells coexpressing alpha-adrenergic (alphaAR) and human fibrinogen (Fg) receptors (integrin alphaIIbbeta3). Stimulation of these cells with alpha-agonists produced a transient rise in the free cytosolic calcium (Ca(++)) accompanied by enhanced binding to soluble Fg, and these effects were prevented by specific alphaAR antagonists. The alpha-adrenergic-induced activation of alphaIIbbeta3 in CHO-alphaIIbbeta3-alphaAR increased the rate of adhesion and extension of cells onto Fg coated plates, and also induced a soluble Fg- and alphaIIbbeta3-dependent formation of cell aggregates, whereas no effects were observed by the stimulation of CHO-alphaIIbbeta3 cells. alpha-Adrenergic antagonists, the ligand mimetic peptide RGDS, pertussis toxin (PTX), or EDTA, they all prevented the alpha-adrenergic stimulation of adhesion and aggregation. However, inhibition of PKC prevented the alpha-adrenergic stimulation of cell adherence, whereas blocking the intracellular Ca(++) mobilization impeded the stimulation of cell aggregation. The alpha-adrenergic activation was associated with phosphorylation of a protein of approximately 100 kDa and proteins of the MAPK family. The former was selectively phosphorylated by alpha-adrenergic stimulation whereas the latter were phosphorylated by the binding of cells to Fg and markedly intensified by alpha-adrenergic stimulation.

Disruption of the Cys5-Cys7 disulfide bridge in the platelet glycoprotein Ibbeta prevents the normal maturation and surface exposure of GPIb-IX complexes.

This work aimed at elucidating the molecular genetic defect in two related patients with Bernard-Soulier syndrome (BSS) phenotype. Flow cytometric analysis revealed undetectable levels of platelet glycoproteins (GP), Ibalpha and IX, although plasma glycocalicin was detectable in both cases. The complete sequencing of GPIbalpha, GPIbbeta, and GPIX revealed the presence of a single point mutation, a G to A substitution, in codon 30 of GPIbbeta, that changes Cys5 to Tyr. The parents and sibling of the patients, heterozygotes for this mutation, were asymptomatic and they all showed a reduced platelet content of GPIbalpha and GPIX. Transient transfection of the mutant GPIbalpha subunit failed to render surface expression of GPIbalpha and exerted a dominant-negative effect on the surface exposure of the GPIb-IX complex. Metabolic labelling and immunoprecipitation analysis of transfected cells indicated that [5Tyr]GPIbbeta may associate with GPIX and GPIbalpha, but the maturation of the GPIb-IX complex is impaired. Substitution of either Cys5 or Cys7 by Ala failed to show surface expression of GPIb-IX, suggesting that the Cys5- Cys7 disulfide loop in GPIbbeta is essential for the efficient processing and trafficking of GPIb-IX complexes toward the plasma membrane. Our findings indicate that the identified novel GPIbbeta mutation is responsible for the BSS phenotype of the patients and provide an explanation for the molecular mechanism underlying the reduced platelet content of GPIb-IX complex in the heterozygous individuals.

A variant thrombasthenic phenotype associated with compound heterozygosity of integrin beta3-subunit: (Met124Val)beta3 alters the subunit dimerization rendering a decreased number of constitutive active alphaIIbbeta3 receptors.

We report the analysis of a variant case of thrombasthenic phenotype that is a compound heterozygote for two mutations located within the metal ion dependent adhesion site (MIDAS) of the beta3 subunit. The patient inherited a maternal allele carrying the Met124Val substitution and a paternal allele that changes Asp119 to Tyr. Phenotyping of the human platelet antigen 1 (HPA-1) showed that the platelet alphaIIbbeta3 complex in the patient was mostly accounted for by the Asp 119Tyr allele that does not bind to fibrinogen (Fg). The patient showed agonistinduced binding of platelets to Fg but neither binding to PAC-1 nor cell aggregation could be detected, most likely due to the minute expression (< or = 5%) of alphaIIb(124Val)beta3 receptors. CHO cells expressing (124Val)beta3 showed a diminished surface expression of alphaIIbbeta3, enhanced adhesion to immobilized Fg, and spontaneous aggregation in the presence of soluble Fg, suggesting that (124Val)beta3 may confer constitutive activity to the alphaIIb(124Val)beta3 receptors. A distinct feature of these cells is the failure of DTT to enhance the binding to soluble Fg and the formation of cell aggregates. The substitution of (124Met)beta3 by either a polar or a positively charged amino acid restored the surface exposure and function of the alphaIIbbeta3 receptors whereas a negatively charged residue did not.

Nonsense mutation in exon-19 of GPIIb associated with thrombasthenic phenotype. Failure of GPIIb(delta597-1008) to form stable complexes with GPIIIa.

We report the molecular genetic analysis of a patient with thrombasthenic phenotype. The lack of surface platelet GPIIb-IIIa complexes and the presence of GPIIIa suggested it was a case of type I Glanzmann's thrombasthenia due to a mutation in GPIIb. Single stranded conformational polymorphism analysis (SSCP) of exon-19 of GPIIb showed polymorphic DNA bands. The DNA sequence of exon-19 revealed the presence of a homozygous C1882T transition that changes residue R597 to STOP codon. Since no other mutations were found in either GPIIb or GPIIIa it is concluded that the C1882T substitution in GPIIb is responsible for the thrombasthenic phenotype of the patient. The lack of platelet GPIIb-mRNA in the proband indicates instability of the [C1882T]GPIIb-mRNA. Coexpression of normal GPIIIa and GPIIb(delta597-1008) in CHO cells failed to show surface expression of GPIIb(delta597-1008)-IIIa complexes. Immunoprecipitation analysis demonstrated that GPIIb(delta597-1008) may indeed complex GPIIIa; however, the association is either unstable or incapable of progressing along the secretory pathway.

Megakaryocyte gene targeting mediated by restricted expression of recombinase Cre.

The availability of mice with tissue-specific expression of recombinase Cre is the limiting step for a successful gene targeting by the Cre-LoxP methodology. This work aimed at generating transgenic mice with restricted expression of recombinase Cre in megakaryocytes and platelets, driven by the promoter of the αIIb gene (mαIIb-cre). Mice oocytes were microinjected with a 4.1 Kb construct comprising a 2.7 Kb promoter fragment of the glycoprotein αIIb gene, linked to the Cre-cDNA and followed by the polyA tail of the SV40. We found four mice with positive DNA genotype and three probable sites of genomic integration of the transgene. Only two of the founders showed presence of Cre-mRNA and production of Cre protein, restricted to megakaryocytes. The activity of Cre in mediating gene targeting was assessed by crossing mαIIb-cre mice to Cre-reporter mice (ROSA26-lacZ). The activity of ß-galactosidase, detected only in megakaryocytes, was sufficient to generate intense staining of X-Gal in hepatic haematopoietic islands of 14.5 dpc fetuses, in bone marrow megakaryocytes and platelets from adult mice as well as in vitro cultured megakaryocytes differentiated from bone marrow hematopoietic stem cells. Moreover, the recombinase activity was sufficient to produce the specific gene targeting of a floxed CD40L allele in megakaryocytes. The mαIIb-cre transgenic mice with restricted production of Cre in megakaryocytes, offers a selective, alternative, new tool for the genetic analysis of platelet pathophysiology.

Diminished thrombogenic responses by deletion of the Podocalyxin Gene in mouse megakaryocytes.

Podocalyxin (Podxl) is a type I membrane sialoprotein of the CD34 family, originally described in the epithelial glomerular cells of the kidney (podocytes) in which it plays an important function. Podxl can also be found in megakaryocytes and platelets among other extrarenal places. The surface exposure of Podxl upon platelet activation suggested it could play some physiological role. To elucidate the function of Podxl in platelets, we generated mice with restricted ablation of the podxl gene in megakaryocytes using the Cre-LoxP gene targeting methodology. Mice with Podxl-null megakaryocytes did not show any apparent phenotypical change and their rates of growth, life span and fertility did not differ from the floxed controls. However, Podxl-null mice showed prolonged bleeding time and decreased platelet aggregation in response to physiological agonists. The number, size-distribution and polyploidy of Podxl-null megakaryocytes were similar to the floxed controls. Podxl-null platelets showed normal content of surface receptors and normal activation by agonists. However, the mice bearing Podxl-null platelets showed a significant retardation in the ferric chloride-induced occlusion of the carotid artery. Moreover, acute thrombosis induced by the i.v. injection of sublethal doses of collagen and phenylephrine produced a smaller fall in the number of circulating platelets in Podxl-null mice than in control mice. In addition, perfusion of uncoagulated blood from Podxl-null mice in parallel flow chamber showed reduced adhesion of platelets and formation of aggregates under high shear stress. It is concluded that platelet Podxl is involved in the control of hemostasis acting as a platelet co-stimulator, likely due to its pro-adhesive properties.

Overexpression of podocalyxin in megakaryocytes and platelets decreases the bleeding time and enhances the agonist-induced aggregation of platelets.

Podocalyxin (PODXL) is a 145KDa sialoprotein abundantly expressed in the glycocalix of the intraglomerular kidney epithelial cells, essential in maintaining a normal renal function. PODXL is also found in vascular endothelial cells, megakaryocytes and platelets. The function of PODXL in platelets is ignored; however, its surface exposure upon platelet activation suggests its participation in controlling the hemostasis. We have generated mice (pralphaIIb-PODXL) overexpressing PODXL specifically in megakaryocytes , either alone or as a fusion protein with green fluorescent protein. The transgenic mice showed a phenotype characterized by decreased bleeding time, mild rebleeding and enhanced platelets aggregation upon agonist stimulation. The cytohematological exams as well as the prothrombin time (PT) and (APTT) tests did not differ from the control group. The biochemical analysis showed only a discrete hyperlipemia and a rise in plasma uric acid levels in the transgenic mice. The present data seem to indicate that PODXL may act as a costimulator of agonists in the activation of platelets and formation of a stable thrombus.

Modification of kidney barrier function by the urokinase receptor.

Podocyte dysfunction, represented by foot process effacement and proteinuria, is often the starting point for progressive kidney disease. Therapies aimed at the cellular level of the disease are currently not available. Here we show that induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via a mechanism that includes lipid-dependent activation of alphavbeta3 integrin. Mice lacking uPAR (Plaur-/-) are protected from lipopolysaccharide (LPS)-mediated proteinuria but develop disease after expression of a constitutively active beta3 integrin. Gene transfer studies reveal a prerequisite for uPAR expression in podocytes, but not in endothelial cells, for the development of LPS-mediated proteinuria. Mechanistically, uPAR is required to activate alphavbeta3 integrin in podocytes, promoting cell motility and activation of the small GTPases Cdc42 and Rac1. Blockade of alphavbeta3 integrin reduces podocyte motility in vitro and lowers proteinuria in mice. Our findings show a physiological role for uPAR signaling in the regulation of kidney permeability.

Agonist-induced aggregation of Chinese hamster ovary cells coexpressing the human receptors for fibrinogen (integrin alphaIIbbeta3) and the platelet-activating factor: dissociation between adhesion and aggregation.

This work reports the establishment of a Chinese hamster ovary (CHO) cell line stably coexpressing the human alphaIIbbeta3 integrin and the platelet-activating factor receptor (PAFR). These cells aggregate in response to PAF in a Ca(++), alphaIIbbeta3, and soluble fibrinogen (Fg)-dependent manner that is prevented by PAF antagonists or alphaIIbbeta3 blockade. The aggregating response is accompanied by enhanced binding of fibrinogen and the activation-dependent IgM PAC1. This model has permitted us to identify, for the first time, intracellular signals distinctly associated with either alphaIIbbeta3-mediated adhesion or aggregation. Nonreceptor activation of protein kinase C (PKC) by phorbol ester produced cellular adhesion and spreading onto immobilized Fg, but it was not a sufficient signal to provoke cellular aggregation. Moreover, inhibition of PKC impeded the PAF stimulation of cellular adhesion, whereas the aggregation was not prevented. The PAF-induced cellular aggregation was distinctly associated with signaling events arising from the liganded Fg receptor and the agonist-induced stimulation of a calcium/calmodulin-dependent signaling pathway. Sustained tyrosine phosphorylation of both mitogen-activated protein kinase (MAPK) and an approximately 100-kd protein was associated with the PAF-induced aggregation, whereas phosphorylation of focal adhesion kinase (FAK) was preferably associated with cellular adherence and spreading onto immobilized Fg.

Disruption of the beta3 663-687 disulfide bridge confers constitutive activity to beta3 integrins.

The platelet fibrinogen receptor, integrin alphaIIbbeta3, is a noncovalent heterodimer of glycoproteins IIb and IIIa. This work was aimed at elucidating the role played by the carboxy-terminal extracellular, trans-membrane, and cytoplasmic regions of the glycoprotein beta3 in the formation of functional complexes with alpha subunits. Progressive carboxy-terminal deletions of beta3 revealed that surface exposure of alphaIIbbeta3 or alphavbeta3 could not occur in the absence of the transmembrane domain of beta3. In contrast, internal deletions 616 to 690 of the carboxy-terminal regions of the beta3 ectodomain led to surface exposure of constitu tive active receptors in CHO cells, as indicated by the enhanced rate of cell adhesion to immobilized ligands and spontaneous binding to soluble fibrinogen or activation-dependent antibody PAC-1. The functional analysis of cysteine mutations within the 616 to 690 region of beta3 or chimeric beta3-beta7 subunits revealed that disruption of the C663-C687 disulfide bridge endows constitutive activity to the alphaIIbbeta3 receptor. It is concluded that the carboxy-terminal tail of the beta3 ectodomain, so-called beta tail domain (betaTD), is not essential for cell surface expression of beta3 receptors. However, a basal, nonactivated, low ligand-affinity state of the beta3 integrins demands a normal conformation of this domain.