Manuel A. Pallero

Thrombospondin Mediates Focal Adhesion Disassembly through Interactions with Cell Surface Calreticulin

Thrombospondin induces reorganization of the actin cytoskeleton and restructuring of focal adhesions. This activity is localized to amino acids 17–35 in the N-terminal heparin-binding domain of thrombospondin and can be replicated by a peptide (hep I) with this sequence. Thrombospondin/hep I stimulate focal adhesion disassembly through a mechanism involving phosphoinositide 3-kinase activation. However, the receptor for this thrombospondin sequence is unknown. We now report that calreticulin on the cell surface mediates focal adhesion disassembly by thrombospondin/hep I. A 60-kDa protein from endothelial cell detergent extracts has homology and immunoreactivity to calreticulin, binds a hep I affinity column, and neutralizes thrombospondin/hep I-mediated focal adhesion disassembly. Calreticulin on the cell surface was confirmed by biotinylation, confocal microscopy, and by fluorescence-activated cell sorting analyses. Thrombospondin and calreticulin potentially bind through the hep I sequence, since thrombospondin-calreticulin complex formation can be blocked specifically by hep I peptide. Antibodies to calreticulin and preincubation of thrombospondin/hep I with glutathioneS-transferase-calreticulin block thrombospondin/hep I-mediated focal adhesion disassembly and phosphoinositide 3-kinase activation, suggesting that calreticulin is a component of the thrombospondin-induced signaling cascade that regulates cytoskeletal organization. These data identify both a novel receptor for the N terminus of thrombospondin and a distinct role for cell surface calreticulin in cell adhesion.

Low density lipoprotein receptor–related protein is a calreticulin coreceptor that signals focal adhesion disassembly

Thrombospondin (TSP) signals focal adhesion disassembly (the intermediate adhesive state) through interactions with cell surface calreticulin (CRT). TSP or a peptide (hep I) of the active site induces focal adhesion disassembly through binding to CRT, which activates phosphoinositide 3-kinase (PI3K) and extracellular signal–related kinase (ERK) through Gαi2 proteins. Because CRT is not a transmembrane protein, it is likely that CRT signals as part of a coreceptor complex. We now show that low density lipoprotein receptor–related protein (LRP) mediates focal adhesion disassembly initiated by TSP binding to CRT. LRP antagonists (antibodies, receptor-associated protein) block hep I/TSP-induced focal adhesion disassembly. LRP is necessary for TSP/hep I signaling because TSP/hep I is unable to stimulate focal adhesion disassembly or ERK or PI3K signaling in fibroblasts deficient in LRP. LRP is important in TSP–CRT signaling, as shown by the ability of hep I to stimulate association of Gαi2 with LRP. The isolated proteins LRP and CRT interact, and LRP and CRT are associated with hep I in molecular complexes extracted from cells. These data establish a mechanism of cell surface CRT signaling through its coreceptor, LRP, and suggest a novel function for LRP in regulating cell adhesion.

Transducible heat shock protein 20 (HSP20) phosphopeptide alters cytoskeletal dynamics

Activation of cyclic nucleotide dependent signaling pathways leads to relaxation of smooth muscle, alterations in the cytoskeleton of cultured cells, and increases in the phosphorylation of HSP20. To determine the effects of phosphorylated HSP20 on the actin cytoskeleton, phosphopeptide analogs of HSP20 were synthesized. These peptides contained 1) the amino acid sequence surrounding the phosphorylation site of HSP20, 2) a phosphoserine, and 3) a protein transduction domain. Treatment of Swiss 3T3 cells with phosphopeptide analogs of HSP20 led to loss of actin stress fibers and focal adhesion complexes as demonstrated by immunocytochemistry, interference reflection microscopy, and biochemical quantitation of globular‐actin. Treatment with phosphopeptide analogs of HSP20 also led to dephosphorylation of the actin depolymerizing protein cofilin. Pull‐down assays demonstrated that 14‐3‐3 proteins associated with phosphopeptide analogs of HSP20 (but not peptide analogs in which the serine was not phosphorylated). The binding of 14‐3‐3 protein to phosphopeptide analogs of HSP20 prevented the association of cofilin with 14‐3‐3. These data suggest that HSP20 may modulate actin cytoskeletal dynamics by competing with the actin depolymerizing protein cofilin for binding to the scaffolding protein 14‐3‐3. Interestingly, the entire protein was not needed for this effect, suggesting that the association is modulated by phosphopeptide motifs of HSP20. These data also suggest the possibility that cyclic nucleotide dependent relaxation of smooth muscle may be mediated by a thin filament (actin) regulatory process. Finally, these data suggest that protein transduction can be used as a tool to elucidate the specific function of peptide motifs of proteins.

The Anti-adhesive Activity of Thrombospondin Is Mediated by the N-terminal Domain of Cell Surface Calreticulin

Thrombospondin (TSP) induces reorganization of the actin cytoskeleton and restructuring of focal adhesions through binding of amino acids (aa) 17–35 (hep I peptide) of thrombospondin to a cell surface form of calreticulin (CRT). In this report we provide further evidence for the involvement of calreticulin in thrombospondin signaling and characterize thrombospondin-calreticulin interactions. Wild type but notcrt −/− cells respond to hep I/TSP. Responsiveness can be restored by incubation of cells with exogenous calreticulin or by transfection with calreticulin. Thrombospondin forms complexes with the CRT-N-domain that are enhanced by physiologic levels of calcium and zinc. Consistent with thrombospondin/CRT-N-domain binding, only the CRT-N-domain blocks hep I- and thrombospondin-stimulated focal adhesion disassembly. A series of glutathione S-transferase-N-domain mutants were used to map the sequence within the N-domain that interacts with TSP/hep I. A construct containing aa 1–43 but not a construct of aa 1–31 supported thrombospondin binding and focal adhesion disassembly. A series of overlapping peptides were used to further map the thrombospondin-binding site. Peptides spanning aa 19–36 (RWIESKHKSDFGKFVLSS) blocked hep I-stimulated focal adhesion disassembly, indicating that the TSP/hep I-binding site is located to this sequence in calreticulin. A mutant fusion protein lacking aa 19–36 (glutathione S-transferase-CRTΔhep I) failed to restore responsiveness to hep I in crt −/−cells, bind thrombospondin, or competitively block focal adhesion disassembly, providing evidence for the role of this calreticulin sequence in mediating thrombospondin signaling.

Thrombospondin Stimulates Focal Adhesion Disassembly through Gi- and Phosphoinositide 3-Kinase-dependent ERK Activation

The matricellular protein thrombospondin (TSP) stimulates stress fiber and focal adhesion disassembly through a sequence (hep I) in its heparin-binding domain. TSP/hep I signals focal adhesion disassembly by binding cell surface calreticulin (CRT) and activating phosphoinositide 3-kinase (PI3K). However, other components of this signaling pathway have not been identified. We now show that TSP induces focal adhesion disassembly through activation of pertussis toxin (PTX)-sensitive G proteins and ERK phosphorylation. PTX pretreatment inhibits TSP/hep I-mediated focal adhesion disassembly as well as PI3K activation. In addition, membrane-permeable Gαi2- and Gβγ-blocking peptides inhibit hep I-mediated focal adhesion disassembly. Hep I stimulates a transient increase in ERK activation, which is abrogated by both PTX and PI3K inhibitors. Inhibiting ERK activation with MEK inhibitors blocks hep I-mediated focal adhesion disassembly, indicating that ERK activation is required for cytoskeletal reorganization. G protein signals and ERK phosphorylation are induced by TSP binding to cell surface CRT, because CRT null mouse embryonic fibroblasts (MEF) fail to stimulate ERK phosphorylation in response to TSP/hep I treatment. These data show that Gi protein and ERK, in concert with PI3K, are stimulated by TSP·CRT interactions at the cell surface to induce de-adhesive changes in the cytoskeleton.

Thrombospondin Signaling of Focal Adhesion Disassembly Requires Activation of Phosphoinositide 3-Kinase

Thrombospondin is an extracellular matrix protein involved in modulating cell adhesion. Thrombospondin stimulates a rapid loss of focal adhesion plaques and reorganization of the actin cytoskeleton in cultured bovine aortic endothelial cells. The focal adhesion labilizing activity of thrombospondin is localized to the amino-terminal domain, specifically amino acids 17–35. Use of a synthetic peptide (hep I), containing amino acids 17–35 of thrombospondin, enables us to examine the signaling mechanisms specifically involved in thrombospondin-induced disassembly of focal adhesions. We tested the hypothesis that activation of phosphoinositide 3-kinase is a necessary step in the thrombospondin-induced signaling pathway regulating focal adhesion disassembly. Both wortmannin and LY294002, membrane permeable inhibitors of phosphoinositide 3-kinase activity, blocked hep I-induced disassembly of focal adhesions. Similarly, wortmannin inhibited hep I-mediated actin microfilament reorganization and the hep I-induced translocation of α-actinin from focal adhesion plaques. Hep I also stimulated phosphoinositide 3-kinase activity approximately 2–3-fold as measured in anti-phosphoinositide 3-kinase and anti-phosphotyrosine immunoprecipitates. Increased immunoreactivity for the 85-kDa regulatory subunit in anti-phosphotyrosine immunoprecipitates suggests that the p85/p110 form of phosphoinositide 3-kinase is involved in this pathway. In32Pi-labeled cells, hep I increased levels of phosphatidylinositol (3,4,5)-trisphosphate, the major product of phosphoinositide 3-kinase phosphorylation. These results suggest that thrombospondin signals the disassembly of focal adhesions and reorganization of the actin cytoskeleton by a pathway involving stimulation of phosphoinositide 3-kinase activity.

Thrombospondin 1 binding to calreticulin-LRP1 signals resistance to anoikis

Anoikis, apoptotic cell death due to loss of cell adhesion, is critical for regulation of tissue homeostasis in tissue remodeling. Fibrogenesis is associated with reduced fibroblast apoptosis. The matricellular protein thrombospondin 1 (TSP1) regulates cell adhesion and motility during tissue remodeling and in fibrogenesis. The N‐terminal domain of TSP1 binds to the calreticulin‐LRP1 receptor co‐complex to signal down‐regulation of cell adhesion and increased cell motility through focal adhesion disassembly. TSP1 signaling through calreticulin‐LRP1 activates cell survival signals such as PI3‐kinase. Therefore, we tested the hypothesis that TSP1 supports cell survival under adhesion‐independent conditions to facilitate tissue remodeling. Here, we show that platelet TSP1, its N‐terminal domain (NoC1) as a recombinant protein, or a peptide comprising the calreticulin‐LRP1 binding site [amino acids 17‐35 (hep I)] in the N‐terminal domain promotes fibroblast survival under anchorage‐independent conditions. TSP1 activates Akt and decreases apoptotic signaling through caspase 3 and PARP1 in suspended fibroblasts. Inhibition of PI3K/Akt activity blocks TSP1‐mediated anchorage‐independent survival. Fibroblasts lacking LRP1 or expressing calreticulin lacking the TSP1 binding site do not respond to TSP1 with anchorage‐independent survival. These data define a novel role for TSP1 signaling through the calreticulin/ LRP1 co‐complex in tissue remodeling and fibrotic responses through stimulation of anoikis resistance.— Pallero, M. A., Elzie, C. A., Chen, J., Mosher, D. F., Murphy‐Ullrich, J. E. Thrombospondin 1 binding to calreticulin‐LRP1 signals resistance to anoikis. FASEB J. 22, 3968–3979 (2008)

Epidermal Growth Factor-like Repeats of Thrombospondins Activate Phospholipase Cγ and Increase Epithelial Cell Migration through Indirect Epidermal Growth Factor Receptor Activation

Thrombospondin (TSP) 1 is a trimeric multidomain protein that contains motifs that recognize distinct host cell receptors coupled to multiple signaling pathways. Selected TSP1-induced cellular responses are tyrosine kinase-dependent, and TSP1 contains epidermal growth factor (EGF)-like repeats. Specific receptor interactions or functions for the EGF-like repeats have not been identified. We asked whether one or more biological responses to TSP1 might be explained through EGF receptor (EGFR) activation. In A431 cells, TSP1 increased autophosphorylation of Tyr-1068 of EGFR in a dose- and time-dependent manner. The ability of TSP1 to activate EGFR was replicated by the tandem EGF-like repeats as a recombinant protein. The three EGF-like repeats alone produced a high level of Tyr-1068 phosphorylation. EGF-like repeats from TSP2 and TSP4 also activated EGFR. Tyr-1068 phosphorylation was less when individual EGF-like repeats were tested or flanking sequences were added to the three EGF-like repeats. TSP1 and its EGF-like repeats also increased phosphorylation of EGFR Tyr-845, Tyr-992, Tyr-1045, Tyr-1086, and Tyr-1173, activated phospholipase Cγ, and increased cell migration. No evidence was found for binding of the EGF-like repeats to EGFR. Instead, EGFR activation in response to TSP1 or its EGF-like repeats required matrix metalloprotease activity, including activity of matrix metalloprotease 9. Access to the ligand-binding portion of the EGFR ectodomain was also required. These findings suggest release of an endogenous EGFR ligand in response to ligation of a second unknown receptor by the TSPs.

Intracellular calreticulin regulates multiple steps in fibrillar collagen expression, trafficking, and processing into the extracellular matrix

Calreticulin (CRT), a chaperone and Ca2+ regulator, enhances wound healing, and its expression correlates with fibrosis in animal models, suggesting that CRT regulates production of the extracellular matrix. However, direct regulation of collagen matrix by CRT has not been previously demonstrated. We investigated the role of CRT in the regulation of fibrillar collagen expression, secretion, processing, and deposition in the extracellular matrix by fibroblasts. Mouse embryonic fibroblasts deficient in CRT (CRT−/− MEFs) have reduced transcript levels of fibrillar collagen I and III and less soluble collagen as compared with wild type MEFs. Correspondingly, fibroblasts engineered to overexpress CRT have increased collagen type I transcript and protein. Collagen expression appears to be regulated by endoplasmic reticulum (ER) calcium levels and intracellular CRT, because thapsigargin treatment reduced collagen expression, whereas addition of exogenous recombinant CRT had no effect. CRT−/− MEFs exhibited increased ER retention of collagen, and collagen and CRT were co-immunoprecipitated from isolated cell lysates, suggesting that CRT is important for trafficking of collagen through the ER. CRT−/− MEFs also have reduced type I procollagen processing and deposition into the extracellular matrix. The reduced collagen matrix deposition is partly a consequence of reduced fibronectin matrix formation in the CRT-deficient cells. Together, these data show that CRT complexes with collagen in cells and that CRT plays critical roles at multiple stages of collagen expression and processing. These data identify CRT as an important regulator of collagen and suggest that intracellular CRT signaling plays an important role in tissue remodeling and fibrosis.

Calreticulin Regulates Transforming Growth Factor-β-stimulated Extracellular Matrix Production

Background: Endoplasmic reticulum (ER) stress is associated with fibrotic diseases, although the mechanisms are not completely understood. Results: The ER stress protein calreticulin regulates TGF-β stimulated extracellular matrix through control of intracellular calcium and NFAT signaling. Conclusion: Calreticulin is necessary for TGF-β stimulated extracellular matrix production. Significance: These findings identify calreticulin as a mechanistic link between ER stress and fibrosis. Endoplasmic reticulum (ER) stress is an emerging factor in fibrotic disease, although precise mechanisms are not clear. Calreticulin (CRT) is an ER chaperone and regulator of Ca2+ signaling up-regulated by ER stress and in fibrotic tissues. Previously, we showed that ER CRT regulates type I collagen transcript, trafficking, secretion, and processing into the extracellular matrix (ECM). To determine the role of CRT in ECM regulation under fibrotic conditions, we asked whether CRT modified cellular responses to the pro-fibrotic cytokine, TGF-β. These studies show that CRT−/− mouse embryonic fibroblasts (MEFs) and rat and human idiopathic pulmonary fibrosis lung fibroblasts with siRNA CRT knockdown had impaired TGF-β stimulation of type I collagen and fibronectin. In contrast, fibroblasts with increased CRT expression had enhanced responses to TGF-β. The lack of CRT does not impact canonical TGF-β signaling as TGF-β was able to stimulate Smad reporter activity in CRT−/− MEFs. CRT regulation of TGF-β-stimulated Ca2+ signaling is important for induction of ECM. CRT−/− MEFs failed to increase intracellular Ca2+ levels in response to TGF-β. NFAT activity is required for ECM stimulation by TGF-β. In CRT−/− MEFs, TGF-β stimulation of NFAT nuclear translocation and reporter activity is impaired. Importantly, CRT is required for TGF-β stimulation of ECM under conditions of ER stress, as tunicamycin-induced ER stress was insufficient to induce ECM production in TGF-β stimulated CRT−/− MEFs. Together, these data identify CRT-regulated Ca2+-dependent pathways as a critical molecular link between ER stress and TGF-β fibrotic signaling.