Calreticulin is important for the development of renal fibrosis and dysfunction in diabetic nephropathy.

Previously, our lab showed that the endoplasmic reticulum (ER) and calcium regulatory protein, calreticulin (CRT), is important for collagen transcription, secretion, and assembly into the extracellular matrix (ECM) and that ER CRT is critical for TGF-β stimulation of type I collagen transcription through stimulation of ER calcium release and NFAT activation. Diabetes is the leading cause of end stage renal disease. TGF-β is a key factor in the pathogenesis of diabetic nephropathy.

Identification of Inhibitors of Thrombospondin 1 Activation of TGF-β.

TGF-β has been a target of interest for the treatment of fibrotic diseases and certain cancers. Approaches to target TGF-β include antagonists of the active ligand or TGF-β receptor kinase activity. These approaches have failed in clinical trials due to a lack of effectiveness and a limited therapeutic window.

Calreticulin Regulates Neointima Formation and Collagen Deposition following Carotid Artery Ligation.

Background/aims: The endoplasmic reticulum (ER) stress protein, calreticulin (CRT), is required for the production of TGF-β-stimulated extracellular matrix (ECM) by fibroblasts. Since TGF-β regulates vascular fibroproliferative responses and collagen deposition, we investigated the effects of CRT knockdown on vascular smooth-muscle cell (VSMC) fibroproliferative responses and collagen deposition.

Methods: Using a carotid artery ligation model of vascular injury, Cre-recombinase-IRES-GFP plasmid was delivered with microbubbles (MB) to CRT-floxed mice using ultrasound (US) to specifically reduce CRT expression in the carotid artery.

Inhibition of Transforming Growth Factor-β Activation Diminishes Tumor Progression and Osteolytic Bone Disease in Mouse Models of Multiple Myeloma.

Transforming growth factor (TGF)-β supports multiple myeloma progression and associated osteolytic bone disease. Conversion of latent TGF-β to its biologically active form is a major regulatory node controlling its activity. Thrombospondin1 (TSP1) binds and activates TGF-β.

Hyperglycemic arterial disturbed flow niche as an in vitro model of atherosclerosis.

Type 2 diabetes significantly elevates the risk of cardiovascular disease. This can be largely attributed to the adverse effects of hyperglycemic conditions on normal endothelial cell (EC) function. ECs in both large and small vessels are influenced by hyperglycemic conditions, which increase susceptibility to EC dysfunction and atherosclerotic lesion formation.

Calreticulin regulates transforming growth factor-β-stimulated extracellular matrix production.

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 Ca(2+) 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).

Thrombospondin-1 opens the paracellular pathway in pulmonary microvascular endothelia through EGFR/ErbB2 activation.

Thrombospondin-1 (TSP1) is a multidomain protein that contains epidermal growth factor (EGF)-like repeats that indirectly activate the EGF receptor (EGFR) and selected downstream signaling pathways. In these studies, we show that TSP1 opens the paracellular pathway in human lung microvascular endothelial cells (HMVEC-Ls) in a dose-, time-, and protein tyrosine kinase (PTK)-dependent manner. TSP1 increased tyrosine phosphorylation of proteins enriched to intercellular boundaries including the zonula adherens (ZA) proteins, vascular endothelial-cadherin, γ-catenin, and p120 catenin.

The calreticulin-binding sequence of thrombospondin 1 regulates collagen expression and organization during tissue remodeling.

Amino acids 17-35 of the thrombospondin1 (TSP1) N-terminal domain (NTD) bind cell surface calreticulin to signal focal adhesion disassembly, cell migration, and anoikis resistance in vitro. However, the in vivo relevance of this signaling pathway has not been previously determined. We engineered local in vivo expression of the TSP1 calreticulin-binding sequence to determine the role of TSP1 in tissue remodeling.

Modulation of mammary cancer cell migration by 15-deoxy-delta(12,14)-prostaglandin J(2): implications for anti-metastatic therapy.

Recently, a number of steps in the progression of metastatic disease have been shown to be regulated by redox signalling. Electrophilic lipids affect redox signalling through the post-translational modification of critical cysteine residues in proteins. However, the therapeutic potential as well as the precise mechanisms of action of electrophilic lipids in cancer cells is poorly understood.

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

Calreticulin (CRT), a chaperone and Ca(2+) 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.

Stainless steel ions stimulate increased thrombospondin-1-dependent TGF-beta activation by vascular smooth muscle cells: implications for in-stent restenosis.

Background/aims: Despite advances in stent design, in-stent restenosis (ISR) remains a significant clinical problem. All implant metals exhibit corrosion, which results in release of metal ions. Stainless steel (SS), a metal alloy widely used in stents, releases ions to the vessel wall and induces reactive oxygen species, inflammation and fibroproliferative responses.

Epidermal growth factor-like repeats of thrombospondins activate phospholipase Cgamma 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.

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 small heat shock-related protein, HSP20, is a cAMP-dependent protein kinase substrate that is involved in airway smooth muscle relaxation.

Activation of the cAMP/cAMP-dependent PKA pathway leads to relaxation of airway smooth muscle (ASM). The purpose of this study was to examine the role of the small heat shock-related protein HSP20 in mediating PKA-dependent ASM relaxation. Human ASM cells were engineered to constitutively express a green fluorescent protein-PKA inhibitory fusion protein (PKI-GFP) or GFP alone.

A thrombospondin-1 antagonist of transforming growth factor-beta activation blocks cardiomyopathy in rats with diabetes and elevated angiotensin II.

In diabetes and hypertension, the induction of increased transforming growth factor-beta (TGF-beta) activity due to glucose and angiotensin II is a significant factor in the development of fibrosis and organ failure. We showed previously that glucose and angiotensin II induce the latent TGF-beta activator thrombospondin-1 (TSP1). Because activation of latent TGF-beta is a major means of regulating TGF-beta, we addressed the role of TSP1-mediated TGF-beta activation in the development of diabetic cardiomyopathy exacerbated by abdominal aortic coarctation in a rat model of type 1 diabetes using a peptide antagonist of TSP1-dependent TGF-beta activation.

Thy-1, via its GPI anchor, modulates Src family kinase and focal adhesion kinase phosphorylation and subcellular localization, and fibroblast migration, in response to thrombospondin-1/hep I.

Normal fibroblast subpopulations have differential surface expression of the GPI-linked raft protein Thy-1, which correlates with differences in cellular adhesion and migration in vitro. Thrombospondin-1 (TSP-1) induces an intermediate state of adhesion in fibroblasts and other cells which facilitates migration. TSP-1 and the hep I peptide derived from the amino-terminal/heparin-binding domain of TSP-1 induce disassembly of cellular focal adhesions.

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.

Thrombospondin induces RhoA inactivation through FAK-dependent signaling to stimulate focal adhesion disassembly.

Cells utilize dynamic interactions with the extracellular matrix to adapt to changing environmental conditions. Thrombospondin 1 (TSP1) induces focal adhesion disassembly and cell migration through a sequence (hep I) in its heparin-binding domain signaling through the calreticulin-low density lipoprotein receptor-related protein receptor complex. This involves the Galphai-dependent activation of ERK and phosphoinositide (PI) 3-kinase, both of which are required for focal adhesion disassembly.

Thrombospondin-1-induced focal adhesion disassembly in fibroblasts requires Thy-1 surface expression, lipid raft integrity, and Src activation.

The hep I peptide of thrombospondin-1 is known to induce the disassembly of focal adhesions, a critical step in regulating cellular adhesive changes needed for cell motility. Fibroblasts that are heterogeneous with respect to the surface expression of Thy-1 differ markedly in morphology, cytoskeletal organization, and migration, suggesting differential regulation of focal adhesion dynamics. Here we demonstrate that disassembly of focal adhesions mediated by both full-length thrombospondin-1 and the hep I peptide in fibroblasts requires the expression of Thy-1, although it does not appear to function as a stable member of the hep I receptor complex.

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 Galphai2 proteins. Because CRT is not a transmembrane protein, it is likely that CRT signals as part of a coreceptor complex.

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 not crt(-/-) cells respond to hep I/TSP.

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.