Philip E. Lapinski

MicroRNA-132–mediated loss of p120RasGAP activates the endothelium to facilitate pathological angiogenesis

Although it is well established that tumors initiate an angiogenic switch, the molecular basis of this process remains incompletely understood. Here we show that the miRNA miR-132 acts as an angiogenic switch by targeting p120RasGAP in the endothelium and thereby inducing neovascularization. We identified miR-132 as a highly upregulated miRNA in a human embryonic stem cell model of vasculogenesis and found that miR-132 was highly expressed in the endothelium of human tumors and hemangiomas but was undetectable in normal endothelium. Ectopic expression of miR-132 in endothelial cells in vitro increased their proliferation and tube-forming capacity, whereas intraocular injection of an antagomir targeting miR-132, anti–miR-132, reduced postnatal retinal vascular development in mice. Among the top-ranking predicted targets of miR-132 was p120RasGAP, which we found to be expressed in normal but not tumor endothelium. Endothelial expression of miR-132 suppressed p120RasGAP expression and increased Ras activity, whereas a miRNA-resistant version of p120RasGAP reversed the vascular response induced by miR-132. Notably, administration of anti–miR-132 inhibited angiogenesis in wild-type mice but not in mice with an inducible deletion of Rasa1 (encoding p120RasGAP). Finally, vessel-targeted nanoparticle delivery of anti–miR-132 restored p120RasGAP expression in the tumor endothelium, suppressed angiogenesis and decreased tumor burden in an orthotopic xenograft mouse model of human breast carcinoma. We conclude that miR-132 acts as an angiogenic switch by suppressing endothelial p120RasGAP expression, leading to Ras activation and the induction of neovascularization, whereas the application of anti–miR-132 inhibits neovascularization by maintaining vessels in the resting state.

Walker A Lysine Mutations of TAP1 and TAP2 Interfere with Peptide Translocation but Not Peptide Binding

We generated mutants of the transporter associated with antigen-processing subunits TAP1 and TAP2 that were altered at the conserved lysine residue in the Walker A motifs of the nucleotide binding domains (NBD). In other ATP binding cassette transporters, mutations of the lysine have been shown to reduce or abrogate the ATP hydrolysis activity and in some cases impair nucleotide binding. Mutants TAP1(K544M) and TAP2(K509M) were expressed in insect cells, and the effects of the mutations on nucleotide binding, peptide binding, and peptide translocation were assessed. The mutant TAP1 subunit is significantly impaired for nucleotide binding relative to wild type TAP1. The identical mutation in TAP2 does not significantly impair nucleotide binding relative to wild type TAP2. Using fluorescence quenching assays to measure the binding of fluorescent peptides, we show that both mutants, in combination with their wild type partners, can bind peptides. Since the mutant TAP1 is significantly impaired for nucleotide binding, these results indicate that nucleotide binding to TAP1 is not a requirement for peptide binding to TAP complexes. Peptide translocation is undetectable for TAP1·TAP2(K509M) complexes, but low levels of translocation are detectable with TAP1(K544M)·TAP2 complexes. These results suggest an impairment in nucleotide hydrolysis by TAP complexes containing either mutant TAP subunit and indicate that the presence of one intact TAP NBD is insufficient for efficient catalysis of peptide translocation. Taken together, these results also suggest the possibility of distinct functions for TAP1 and TAP2 NBD during a single translocation cycle.

RASA1 maintains the lymphatic vasculature in a quiescent functional state in mice

RASA1 (also known as p120 RasGAP) is a Ras GTPase-activating protein that functions as a regulator of blood vessel growth in adult mice and humans. In humans, RASA1 mutations cause capillary malformation-arteriovenous malformation (CM-AVM); whether it also functions as a regulator of the lymphatic vasculature is unknown. We investigated this issue using mice in which Rasa1 could be inducibly deleted by administration of tamoxifen. Systemic loss of RASA1 resulted in a lymphatic vessel disorder characterized by extensive lymphatic vessel hyperplasia and leakage and early lethality caused by chylothorax (lymphatic fluid accumulation in the pleural cavity). Lymphatic vessel hyperplasia was a consequence of increased proliferation of lymphatic endothelial cells (LECs) and was also observed in mice in which induced deletion of Rasa1 was restricted to LECs. RASA1-deficient LECs showed evidence of constitutive activation of Ras in situ. Furthermore, in isolated RASA1-deficient LECs, activation of the Ras signaling pathway was prolonged and cellular proliferation was enhanced after ligand binding to different growth factor receptors, including VEGFR-3. Blockade of VEGFR-3 was sufficient to inhibit the development of lymphatic vessel hyperplasia after loss of RASA1 in vivo. These findings reveal a role for RASA1 as a physiological negative regulator of LEC growth that maintains the lymphatic vasculature in a quiescent functional state through its ability to inhibit Ras signal transduction initiated through LEC-expressed growth factor receptors such as VEGFR-3.

Lymphatic abnormalities are associated with RASA1 gene mutations in mouse and man

Mutations in gene RASA1 have been historically associated with capillary malformation–arteriovenous malformation, but sporadic reports of lymphatic involvement have yet to be investigated in detail. To investigate the impact of RASA1 mutations in the lymphatic system, we performed investigational near-infrared fluorescence lymphatic imaging and confirmatory radiographic lymphangiography in a Parkes–Weber syndrome (PKWS) patient with suspected RASA1 mutations and correlated the lymphatic abnormalities against that imaged in an inducible Rasa1 knockout mouse. Whole-exome sequencing (WES) analysis and validation by Sanger sequencing of DNA from the patient and unaffected biological parents enabled us to identify an early-frameshift deletion in RASA1 that was shared with the father, who possessed a capillary stain but otherwise no overt disease phenotype. Abnormal lymphatic vasculature was imaged in both affected and unaffected legs of the PKWS subject that transported injected indocyanine green dye to the inguinal lymph node and drained atypically into the abdomen and into dermal lymphocele-like vesicles on the groin. Dermal lymphatic hyperplasia and dilated vessels were observed in Rasa1-deficient mice, with subsequent development of chylous ascites. WES analyses did not identify potential gene modifiers that could explain the variability of penetrance between father and son. Nonetheless, we conclude that the RASA1 mutation is responsible for the aberrant lymphatic architecture and functional abnormalities, as visualized in the PKWS subject and in the animal model. Our unique method to combine investigatory near-infrared fluorescence lymphatic imaging and WES for accurate phenoptyping and unbiased genotyping allows the study of molecular mechanisms of lymphatic involvement of hemovascular disorders.

Tapasin Interacts with the Membrane-spanning Domains of Both TAP Subunits and Enhances the Structural Stability of TAP1·TAP2 Complexes

The transporter associated with antigen processing (TAP) proteins are involved in transport of peptides from the cytosol into the endoplasmic reticulum. Two subunits, TAP1 and TAP2, are necessary and sufficient for peptide binding and peptide translocation across the endoplasmic reticulum membrane. TAP1 and TAP2 contain an N-terminal hydrophobic membrane-spanning region and a C-terminal nucleotide binding domain. Tapasin is an endoplasmic reticulum resident protein that has been found associated with the TAP subunits and shown to increase expression levels of TAP. Here we investigated TAP-tapasin interactions and their effects on TAP function in insect cells. We show tapasin binding to both TAP1 and TAP2 and to the corresponding nucleotide binding domain-exchanged chimeras as well as to a truncated TAP1·TAP2 complex containing just the membrane-spanning regions of TAP1 and TAP2. However, tapasin interactions with either the truncated TAP construct containing just the nucleotide binding domain are not observed. Tapasin is not required for high affinity peptide binding to TAP1·TAP2 complexes, and in fact, the presence of tapasin slightly reduces the affinity of TAP complexes for peptides. However, at near physiological temperatures, both tapasin and nucleotides stabilize the peptide binding site of TAP1·TAP2 complexes against inactivation, and enhanced thermostability of both TAP subunits is observed in the presence of tapasin. The enhanced structural stability of TAP1·TAP2 complexes in the presence of tapasin might explain the observations that tapasin increases TAP protein expression levels in mammalian cells.

Essential role of the T cell–specific adapter protein in the activation of LCK in peripheral T cells

T cell–specific adapter protein (TSAd) is a SRC-homology-2 (SH2) domain–containing intracellular signaling molecule that is required for T cell antigen receptor (TCR)–induced cytokine synthesis in T cells. How TSAd functions in TCR signal transduction is not clear. Previous work has suggested a nuclear role for this adapter. However, other evidence suggests that TSAd also functions in the cytoplasm. Using T cells from TSAd-deficient mice, we now show that the major role of TSAd in the cytoplasm is in activation of the LCK protein tyrosine kinase at the outset of TCR signal transduction. Consequently, TSAd regulates several downstream signaling events, including intracellular calcium mobilization and activation of the Ras–extracellular signal–regulated kinase signaling pathway. TSAd regulates LCK activity directly through physical interaction with LCK SH3 and SH2 domains. These studies reveal TSAd as a positive regulator of proximal TCR signal transduction and provide important new information on the mechanism of TCR-induced LCK activation.

Development of severe skeletal defects in induced SHP-2-deficient adult mice: A model of skeletal malformation in humans with SHP-2 mutations

SUMMARY SHP-2 (encoded by PTPN11) is a ubiquitously expressed protein tyrosine phosphatase required for signal transduction by multiple different cell surface receptors. Humans with germline SHP-2 mutations develop Noonan syndrome or LEOPARD syndrome, which are characterized by cardiovascular, neurological and skeletal abnormalities. To study how SHP-2 regulates tissue homeostasis in normal adults, we used a conditional SHP-2 mouse mutant in which loss of expression of SHP-2 was induced in multiple tissues in response to drug administration. Induced deletion of SHP-2 resulted in impaired hematopoiesis, weight loss and lethality. Most strikingly, induced SHP-2-deficient mice developed severe skeletal abnormalities, including kyphoses and scolioses of the spine. Skeletal malformations were associated with alterations in cartilage and a marked increase in trabecular bone mass. Osteoclasts were essentially absent from the bones of SHP-2-deficient mice, thus accounting for the osteopetrotic phenotype. Studies in vitro revealed that osteoclastogenesis that was stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL) was defective in SHP-2-deficient mice. At least in part, this was explained by a requirement for SHP-2 in M-CSF-induced activation of the pro-survival protein kinase AKT in hematopoietic precursor cells. These findings illustrate an essential role for SHP-2 in skeletal growth and remodeling in adults, and reveal some of the cellular and molecular mechanisms involved. The model is predicted to be of further use in understanding how SHP-2 regulates skeletal morphogenesis, which could lead to the development of novel therapies for the treatment of skeletal malformations in human patients with SHP-2 mutations.

Use of chimeric proteins to investigate the role of transporter associated with antigen processing (TAP) structural domains in peptide binding and translocation

The transporter associated with antigen processing (TAP) comprises two subunits, TAP1 and TAP2, each containing a hydrophobic membrane-spanning region (MSR) and a nucleotide binding domain (NBD). The TAP1/TAP2 complex is required for peptide translocation across the endoplasmic reticulum membrane. To understand the role of each structural unit of the TAP1/TAP2 complex, we generated two chimeras containing TAP1 MSR and TAP2 NBD (T1MT2C) or TAP2 MSR and TAP1 NBD (T2MT1C). We show that TAP1/T2MT1C, TAP2/T1MT2C, and T1MT2C/T2MT1C complexes bind peptide with an affinity comparable to wild-type complexes. By contrast, TAP1/T1MT2C and TAP2/T2MT1C complexes, although observed, are impaired for peptide binding. Thus, the MSRs of both TAP1 and TAP2 are required for binding peptide. However, neither NBD contains unique determinants required for peptide binding. The NBD-switched complexes, T1MT2C/T2MT1C, TAP1/T2MT1C, and TAP2/T1MT2C, all translocate peptides, but with progressively reduced efficiencies relative to the TAP1/TAP2 complex. These results indicate that both nucleotide binding sites are catalytically active and support an alternating catalytic sites model for the TAP transport cycle, similar to that proposed for P-glycoprotein. The enhanced translocation efficiency of TAP1/T2MT1C relative to TAP2/T1MT2C complexes correlates with enhanced binding of the TAP1 NBD-containing constructs to ATP-agarose beads. Preferential ATP interaction with TAP1, if occurring in vivo, might polarize the transport cycle such that ATP binding to TAP1 initiates the cycle. However, our observations that TAP complexes containing two identical TAP NBDs can mediate translocation indicate that distinct properties of the nucleotide binding site per se are not essential for the TAP catalytic cycle.

Nonredundant functions for Ras GTPase-activating proteins in tissue homeostasis.

Mutations in Ras guanosine triphophatase–activating proteins (RasGAPs) cause disease in humans. Inactivation of the small guanosine triphosphate–binding protein Ras during receptor signal transduction is mediated by Ras guanosine triphosphatase (GTPase)–activating proteins (RasGAPs). Ten different RasGAPs have been identified and have overlapping patterns of tissue distribution. However, genetic analyses are revealing critical nonredundant functions for each RasGAP in tissue homeostasis and as regulators of disease processes in mouse and man. Here, we discuss advances in understanding the role of RasGAPs in the maintenance of tissue integrity. Many cell surface receptors trigger activation of the small guanosine triphosphate–binding protein Ras, conveying signals for cell proliferation, survival, and differentiation. However, excessive Ras activation results in uncontrolled tissue growth and susceptibility to diseases, such as cancer. Ras guanosine triphophatase (GTPase)–activating proteins (RasGAPs) inactivate Ras to maintain normal signaling activity. This Review, which has 2 figures, 2 tables, and 147 references, discusses the role of individual RasGAP family members in normal tissue and in disease.

Calreticulin recognizes misfolded HLA-A2 heavy chains

Our studies investigated functional interactions between calreticulin, an endoplasmic reticulum chaperone, and major histocompatibility complex (MHC) class I molecules. Using in vitro thermal aggregation assays, we established that calreticulin can inhibit heat-induced aggregation of soluble, peptide-deficient HLA-A2 purified from supernatants of insect cells. The presence of HLA-A2-specific peptides also inhibits heat-induced aggregation. Inhibition of heat-induced aggregation of peptide-deficient HLA-A2 by calreticulin correlates with a rescue of the HLA-A2 heavy chain from precipitation, by forming high-molecular-weight complexes with calreticulin. Complex formation between HLA-A2 heavy chains and calreticulin occurs at 50°C but not 37°C, suggesting polypeptide-based interactions between the HLA-A2 heavy chain and calreticulin. Once complexes are formed, the addition of peptide is not sufficient to trigger efficient assembly of heavy chain/β2m/peptide complexes. Using a fluorescent peptide-based binding assay, we show that calreticulin does not enhance peptide binding by HLA-A2 at 37°C. We also show that calreticulin itself is converted to oligomeric species on exposure to 37°C or higher temperatures, and that oligomeric forms of calreticulin are active in inhibiting thermal aggregation of peptide-deficient HLA-A2. Taken together, these results suggest that calreticulin functions in the recognition of misfolded MHC class I heavy chains in the endoplasmic reticulum. However, in the absence of other endoplasmic reticulum components, calreticulin by itself does not enhance the assembly of misfolded MHC class I heavy chains with β2m and peptides.