Thrombospondin-1 Signaling Through the Calreticulin/LDL Receptor Related Protein 1 Axis: Functions and Possible Roles in Glaucoma.

Thrombospondin-1 (TSP-1) is a matricellular extracellular matrix protein. Matricellular proteins are components of the extracellular matrix (ECM) that regulate key cellular functions and impact ECM organization, but which lack direct primary structural roles in the ECM. TSP-1 expression is upregulated in response to injury, hypoxia, growth factor stimulation, inflammation, glucose, and by reactive oxygen species. Relevant to glaucoma, TSP-1 is also a mechanosensitive molecule upregulated by mechanical stretch. TSP-1 expression is increased in ocular remodeling in glaucoma in both the trabecular meshwork and in the optic nerve head. The exact roles of TSP-1 in glaucoma remain to be defined, however. It plays important roles in cell behavior and in ECM remodeling during wound healing, fibrosis, angiogenesis, and in tumorigenesis and metastasis. At the cellular level, TSP-1 can modulate cell adhesion and migration, protease activity, growth factor activity, anoikis resistance, apoptosis, and collagen secretion and matrix assembly and cross-linking. These multiple functions and macromolecular and receptor interactions have been ascribed to specific domains of the TSP-1 molecule. In this review, we will focus on the cell regulatory activities of the TSP-1 N-terminal domain (NTD) sequence that binds to cell surface calreticulin (Calr) and which regulates cell functions via signaling through Calr complexed with LDL receptor related protein 1 (LRP1). We will describe TSP-1 actions mediated through the Calr/LRP1 complex in regulating focal adhesion disassembly and cytoskeletal reorganization, cell motility, anoikis resistance, and induction of collagen secretion and matrix deposition. Finally, we will consider the relevance of these TSP-1 functions to the pathologic remodeling of the ECM in glaucoma.

Runx2 Deficiency in Osteoblasts Promotes Myeloma Resistance to Bortezomib by Increasing TSP-1-Dependent TGFß1 Activation and Suppressing Immunity in Bone Marrow.

Multiple myeloma is a plasma cell malignancy that thrives in the bone marrow (BM). The proteasome inhibitor bortezomib is one of the most effective first-line chemotherapeutic drugs for multiple myeloma; however, 15% to 20% of high-risk patients do not respond to or become resistant to this drug and the mechanisms of chemoresistance remain unclear. We previously demonstrated that multiple myeloma cells inhibit Runt-related transcription factor 2 (Runx2) in pre- and immature osteoblasts (OB), and that this OB-Runx2 deficiency induces a cytokine-rich and immunosuppressive microenvironment in the BM. In the current study, we assessed the impact of OB-Runx2 deficiency on the outcome of bortezomib treatment using OB-Runx2+/+ and OB-Runx2-/- mouse models of multiple myeloma. In vitro and in vivo experiments revealed that OB-Runx2 deficiency induces multiple myeloma cell resistance to bortezomib via the upregulation of immunosuppressive myeloid-derived suppressor cells (MDSCs), downregulation of cytotoxic T cells, and activation of TGFß1 in the BM. In multiple myeloma tumor-bearing OB-Runx2-/- mice, treatment with SRI31277, an antagonist of thrombospondin-1 (TSP-1)-mediated TGFß1 activation, reversed the BM immunosuppression and significantly reduced tumor burden. Furthermore, treatment with SRI31277 combined with bortezomib alleviated multiple myeloma cell resistance to bortezomib-induced apoptosis caused by OB-Runx2 deficiency in cocultured cells and produced a synergistic effect on tumor burden in OB-Runx2-/- mice. Depletion of MDSCs by 5-fluorouracil or gemcitabine similarly reversed the immunosuppressive effects and bortezomib resistance induced by OB-Runx2 deficiency in tumor-bearing mice, indicating the importance of the immune environment for drug resistance and suggesting new strategies to overcome bortezomib resistance in the treatment of multiple myeloma.

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

TGF-ß has been a target of interest for the treatment of fibrotic diseases and certain cancers. Approaches to target TGF-ß include antagonists of the active ligand or TGF-ß receptor kinase activity. These approaches have failed in clinical trials due to a lack of effectiveness and a limited therapeutic window. In this context, newer and more selective approaches to target TGF-ß are needed. We previously reported that the matricellular protein, thrombospondin 1, activates the latent TGF-ß complex and that antagonism of this pathway using tri/tetrapeptides in various animal models reduces fibrosis. The tripeptide, SRI-31277 (1), is effective in vivo but has a short plasma half life (0.2 h). Herein we describe the design and synthesis SRI-31277 analogs, specifically smaller peptides that retain potency and have improved bioavailability. We identified SRI-35241 (36) with a single chiral center, which blocks TGF-ß activation (pIC50 = 8.12 nM) and has a plasma half life of 1.8 h (iv).

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-ß 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-ß is a key factor in the pathogenesis of diabetic nephropathy. However, the role of calreticulin (Calr) in fibrosis of diabetic nephropathy has not been investigated. In current work, we used both in vitro and in vivo approaches to assess the role of ER CRT in TGF-ß and glucose stimulated ECM production by renal tubule cells and in diabetic mice. Knockdown of CALR by siRNA in a human proximal tubular cell line (HK-2) showed reduced induction of soluble collagen when stimulated by TGF-ß or high glucose as compared to control cells, as well as a reduction in fibronectin and collagen IV transcript levels. CRT protein is increased in kidneys of mice made diabetic with streptozotocin and subjected to uninephrectomy to accelerate renal tubular injury as compared to controls. We used renal-targeted ultrasound delivery of Cre-recombinase plasmid to knockdown specifically CRT expression in the remaining kidney of uninephrectomized Calr fl/fl mice with streptozotocin-induced diabetes. This approach reduced CRT expression in the kidney, primarily in the tubular epithelium, by 30-55%, which persisted over the course of the studies. Renal function as measured by the urinary albumin/creatinine ratio was improved in the mice with knockdown of CRT as compared to diabetic mice injected with saline or subjected to ultrasound and injected with control GFP plasmid. PAS staining of kidneys and immunohistochemical analyses of collagen types I and IV show reduced glomerular and tubulointerstitial fibrosis. Renal sections from diabetic mice with CRT knockdown showed reduced nuclear NFAT in renal tubules and treatment of diabetic mice with 11R-VIVIT, an NFAT inhibitor, reduced proteinuria and renal fibrosis. These studies identify ER CRT as an important regulator of TGF-ß stimulated ECM production in the diabetic kidney, potentially through regulation of NFAT-dependent ECM transcription.

Thrombospondin 1 and Its Diverse Roles as a Regulator of Extracellular Matrix in Fibrotic Disease.

Thrombospondin 1 (TSP1) is a matricellular extracellular matrix protein that has diverse roles in regulating cellular processes important for the pathogenesis of fibrotic diseases. We will present evidence for the importance of TSP1 control of latent transforming growth factor beta activation in renal fibrosis with an emphasis on diabetic nephropathy. Other functions of TSP1 that affect renal fibrosis, including regulation of inflammation and capillary density, will be addressed. Emerging roles for TSP1 N-terminal domain regulation of collagen matrix assembly, direct effects of TSP1-collagen binding, and intracellular functions of TSP1 in mediating endoplasmic reticulum stress responses in extracellular matrix remodeling and fibrosis, which could potentially affect renal fibrogenesis, will also be discussed. Finally, we will address possible strategies for targeting TSP1 functions to treat fibrotic renal disease.

Denervation-induced skeletal muscle fibrosis is mediated by CTGF/CCN2 independently of TGF-ß.

Muscular fibrosis is caused by excessive accumulation of extracellular matrix (ECM) that replaces functional tissue, and it is a feature of several myopathies and neuropathies. Knowledge of the biology and regulation of pro-fibrotic factors is critical for the development of new therapeutic strategies. Upon unilateral sciatic nerve transection, we observed accumulation of ECM proteins such as collagen and fibronectin in the denervated hindlimb, together with increased levels of the profibrotic factors transforming growth factor type ß (TGF-ß) and connective tissue growth factor (CTGF/CCN2). In mice hemizygous for CTGF/CCN2 or in mice treated with a blocking antibody against CTGF/CCN2, we observed reduced accumulation of ECM proteins after denervation as compared to control mice, with no changes in fibro/adipogenic progenitors (FAPs), suggesting a direct role of CTGF/CCN2 on denervation-induced fibrosis. During time course experiments, we observed that ECM proteins and CTGF/CCN2 levels are increased early after denervation (2-4 days), while TGF-ß signaling shows a delayed kinetics of appearance (1-2 weeks). Furthermore, blockade of TGF-ß signaling does not decrease fibronectin or CTGF levels after 4 days of denervation. These results suggest that in our model CTGF/CCN2 is not up-regulated by canonical TGF-ß signaling early after denervation and that other factors are likely involved in the early fibrotic response following skeletal muscle denervation.

Multiscale simulation of the interaction of calreticulin-thrombospondin-1 complex with a model membrane microdomain.

Cell surface calreticulin (CRT) binding to thrombospondin-1 (TSP1), regulates cell adhesion, migration, anoikis resistance, and collagen production. Due to the essential role of membrane microdomains in CRT-mediated focal adhesion disassembly, we previously studied the effect of raft-like bilayers on TSP1-CRT interactions with all-atom molecular dynamics (AAMD) simulations. However, the simulated systems of protein on the surface of the bilayer(s) in the explicit solvent are too large for long timescale AAMD simulations due to computational expense. In this study, we adopted a multiscale modeling approach of combining AAMD, coarse-grained molecule dynamics (CGMD), and reversed AAMD (REV AAMD) simulations to investigate the interactions of single CRT or of the TSP1-CRT complex with a membrane microdomain at microsecond timescale. Results showed that CRT conformational stabilization by binding of TSP1 in AAMD simulation was undetectable in CGMD simulation, but it was recovered in REV AAMD simulation. Similarly, interactions of the CRT N-domain and TSP1 with the membrane microdomain were lost in CGMD simulations but they were re-gained in the REV AAMD simulations. There was the higher coordination of the CRT P-domain in the TSP1-CRT complex with the lipid components of membrane microdomain compared to that of single CRT, which could directly affect the conformation of CRT and further mediate CRT recruitment of LDL receptor-related protein for signaling events. This study provides structural and molecular insights into TSP1-CRT interactions in a membrane microdomain environment and demonstrates the feasibility of using multiscale simulations to investigate the interactions between protein and membrane microdomains at a long timescale.

Pericytes Elicit Resistance to Vemurafenib and Sorafenib Therapy in Thyroid Carcinoma via the TSP-1/TGFß1 Axis.

PURPOSE: The BRAFV600E oncogene modulates the papillary thyroid carcinoma (PTC) microenvironment, in which pericytes are critical regulators of tyrosine-kinase (TK)-dependent signaling pathways. Although BRAFV600E and TK inhibitors are available, their efficacy as bimodal therapeutic agents in BRAFV600E-PTC is still unknown. EXPERIMENTAL DESIGN: We assessed the effects of vemurafenib (BRAFV600E inhibitor) and sorafenib (TKI) as single agents or in combination in BRAFWT/V600E-PTC and BRAFWT/WT cells using cell-autonomous, pericyte coculture, and an orthotopic mouse model. We also used BRAFWT/V600E-PTC and BRAFWT/WT-PTC clinical samples to identify differentially expressed genes fundamental to tumor microenvironment. RESULTS: Combined therapy blocks tumor cell proliferation, increases cell death, and decreases motility via BRAFV600E inhibition in thyroid tumor cells in vitro. Vemurafenib produces cytostatic effects in orthotopic tumors, whereas combined therapy (likely reflecting sorafenib activity) generates biological fluctuations with tumor inhibition alternating with tumor growth. We demonstrate that pericytes secrete TSP-1 and TGFß1, and induce the rebound of pERK1/2, pAKT and pSMAD3 levels to overcome the inhibitory effects of the targeted therapy in PTC cells. This leads to increased BRAFV600E-PTC cell survival and cell death refractoriness. We find that BRAFWT/V600E-PTC clinical samples are enriched in pericytes, and TSP1 and TGFß1 expression evoke gene-regulatory networks and pathways (TGFß signaling, metastasis, tumor growth, tumor microenvironment/ECM remodeling functions, inflammation, VEGF ligand-VEGF receptor interactions, immune modulation, etc.) in the microenvironment essential for BRAFWT/V600E-PTC cell survival. Critically, antagonism of the TSP-1/TGFß1 axis reduces tumor cell growth and overcomes drug resistance. CONCLUSIONS: Pericytes shield BRAFV600E-PTC cells from targeted therapy via TSP-1 and TGFß1, suggesting this axis as a new therapeutic target for overcoming resistance to BRAFV600E and TK inhibitors.

Thrombospondins: Purification of human platelet thrombospondin-1.

Thrombospondins are a family of five secreted proteins that have diverse roles in modulating cellular function. Thrombospondins-1 and 2 were identified as matricellular proteins based on their functional roles combined with their transient appearance or accumulation in extracellular matrix at specific times during development and in response to injury or stress in mature tissues. Thrombospondin-1 is a major component of platelet α-granules, which provides a convenient source for purification of the protein. Methods are described to prepare thrombospondin-1 from human platelets in a biologically active form with minimal degradation or contamination with other platelet proteins. A nondenaturing method is described for removing bound transforming growth factor-ß1.

The role of the endoplasmic reticulum protein calreticulin in mediating TGF-ß-stimulated extracellular matrix production in fibrotic disease.

Endoplasmic reticulum (ER) stress is a key factor contributing to fibrotic disease. Although ER stress is a short-term adaptive response, with chronic stimulation, it can activate pathways leading to fibrosis. ER stress can induce TGF-ß signaling, a central driver of extracellular matrix production in fibrosis. This review will discuss the role of an ER protein, calreticulin (CRT), which has both chaperone and calcium regulatory functions, in fibrosis. CRT expression is upregulated in multiple different fibrotic diseases. The roles of CRT in regulation of fibronectin extracellular matrix assembly, extracellular matrix transcription, and collagen secretion and processing into the extracellular matrix will be discussed. Evidence for the importance of CRT in ER calcium release and NFAT activation downstream of TGF-ß signaling will be presented. Finally, we will summarize evidence from animal models in which CRT expression is genetically reduced or experimentally downregulated in targeted tissues of adult animals and discuss how these models define a key role for CRT in fibrotic diseases.

Thrombospondin-1 regulation of latent TGF-ß activation: A therapeutic target for fibrotic disease.

Transforming growth factor-ß (TGF-ß) is a central player in fibrotic disease. Clinical trials with global inhibitors of TGF-ß have been disappointing, suggesting that a more targeted approach is warranted. Conversion of the latent precursor to the biologically active form of TGF-ß represents a novel approach to selectively modulating TGF-ß in disease, as mechanisms employed to activate latent TGF-ß are typically cell, tissue, and/or disease specific. In this review, we will discuss the role of the matricellular protein, thrombospondin 1 (TSP-1), in regulation of latent TGF-ß activation and the use of an antagonist of TSP-1 mediated TGF-ß activation in a number of diverse fibrotic diseases. In particular, we will discuss the TSP-1/TGF-ß pathway in fibrotic complications of diabetes, liver fibrosis, and in multiple myeloma. We will also discuss emerging evidence for a role for TSP-1 in arterial remodeling, biomechanical modulation of TGF-ß activity, and in immune dysfunction. As TSP-1 expression is upregulated by factors induced in fibrotic disease, targeting the TSP-1/TGF-ß pathway potentially represents a more selective approach to controlling TGF-ß activity in disease.

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

Transforming growth factor (TGF)-ß supports multiple myeloma progression and associated osteolytic bone disease. Conversion of latent TGF-ß to its biologically active form is a major regulatory node controlling its activity. Thrombospondin1 (TSP1) binds and activates TGF-ß. TSP1 is increased in myeloma, and TSP1-TGF-ß activation inhibits osteoblast differentiation. We hypothesized that TSP1 regulates TGF-ß activity in myeloma and that antagonism of the TSP1-TGF-ß axis inhibits myeloma progression. Antagonists (LSKL peptide, SRI31277) derived from the LSKL sequence of latent TGF-ß that block TSP1-TGF-ß activation were used to determine the role of the TSP1-TGF-ß pathway in mouse models of myeloma. TSP1 binds to human myeloma cells and activates TGF-ß produced by cultured human and mouse myeloma cell lines. Antagonists delivered via osmotic pump in an intratibial severe combined immunodeficiency CAG myeloma model or in a systemic severe combined immunodeficiency CAG-heparanase model of aggressive myeloma reduced TGF-ß signaling (phospho-Smad 2) in bone sections, tumor burden, mouse IL-6, and osteoclasts, increased osteoblast number, and inhibited bone destruction as measured by microcomputed tomography. SRI31277 reduced tumor burden in the immune competent 5TGM1 myeloma model. SRI31277 was as effective as dexamethasone or bortezomib, and SRI31277 combined with bortezomib showed greater tumor reduction than either agent alone. These studies validate TSP1-regulated TGF-ß activation as a therapeutic strategy for targeted inhibition of TGF-ß in myeloma.

High-Magnitude and/or High-Frequency Mechanical Strain Promotes Peripapillary Scleral Myofibroblast Differentiation.

PURPOSE: To determine the effects of altered mechanical strain on human peripapillary scleral (ppSc) fibroblast-to-myofibroblast differentiation. METHODS: Eight human ppSc fibroblast cultures were isolated from three paired eyes and two unilateral eyes of five donors using an explant approach. Human ppSc fibroblast isolates were subjected to 1% and 4% cyclic strain at 0.05 to 5 Hz for 24 hours. Levels of α smooth muscle actin (αSMA) mRNA and protein were determined by real-time PCR and immunoblot. Incorporation of αSMA into actin stress fibers was evaluated by confocal immunofluorescent microscopy. Myofibroblast contractility was measured by fibroblast-populated three-dimensional collagen gel contraction assay and phosphorylation of myosin light chain (MLC20). RESULTS: Human ppSc fibroblasts contained 6% to 47% fully differentiated myofibroblasts before strain application; 4% cyclic strain increased αSMA mRNA and protein expression in ppSc fibroblasts compared with 1% strain applied at 5 Hz, but not at lower frequencies. Seven of eight ppSc fibroblast isolates responded to high-magnitude and high-frequency strain with increased cellular contractility and increased MLC20 phosphorylation. In addition, increasing strain frequency promoted αSMA expression in ppSc fibroblasts under both 1% and 4% strain conditions. CONCLUSIONS: High-magnitude and/or high-frequency mechanical strain promotes differentiation of human ppSc fibroblasts into contractile myofibroblasts, a fibroblast phenotypic change known to be key to tissue injury-repair responses. These findings suggest that the cellular constituent of ppSc may play an important role in the regulation of optic nerve head biomechanics in response to injurious IOP fluctuations.

The Thrombospondin1-TGF-ß Pathway and Glaucoma.

Glaucoma is characterized by abnormal remodeling of the extracellular matrix (ECM) in the trabecular meshwork and in the connective tissue beams of the lamina cribrosa (LC) at the optic nerve head (ONH), which is associated with axonal damage. Mechanical strain can stimulate ECM remodeling and increased expression of matricellular proteins. Thrombospondins 1 and 2 are induced by cyclic mechanical strain in the eye in both the trabecular meshwork and in the LC region of the ONH. TGF-betas 1 and 2 are increased in glaucoma and play a role in the pathologic remodeling of the ECM in the eye in glaucoma. In this study, we address the role of thrombospondin1 as a regulator of latent TGF-beta activation and discuss the potential therapeutic use of antagonists of the thrombospondin1-TGF-beta pathway for treatment of glaucoma.

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-ß-stimulated extracellular matrix (ECM) by fibroblasts. Since TGF-ß 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. RESULTS: In vitro, Cre-recombinase-mediated CRT knockdown in isolated, floxed VSMCs decreased the CRT transcript and protein, and attenuated the induction of collagen I protein in response to TGF-ß. TGF-ß stimulation of collagen I was partly blocked by the NFAT inhibitor 11R-VIVIT. Following carotid artery ligation, CRT staining was upregulated with enhanced expression in the neointima 14-21 days after injury. Furthermore, Cre-recombinase-IRES-GFP plasmid delivered by targeted US reduced CRT expression in the neointima of CRT-floxed mice and led to a significant reduction in neointima formation and collagen deposition. The neointimal cell number was also reduced in mice, with a local, tissue-specific knockdown of CRT. CONCLUSIONS: This work establishes a novel role for CRT in mediating VSMC responses to injury through the regulation of collagen deposition and neointima formation.

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. Fluid shear stress and flow patterns play an essential role in atherogenesis: lesions form only at locations where fluid flow behavior can be classified as "disturbed flow" (i.e., low shear stress recirculation and/or retrograde flow). Since regions of disturbed flow are the focal points of atherosclerotic cardiovascular disease, we hypothesized that the combinatorial effects of high glucose and disturbed flow conditions elicit significantly different responses from ECs than high glucose alone. To validate our hypothesis, we used our endothelial cell culture model (ECCM) to establish vascular niches associated with "normal" and "disturbed" flow conditions typically seen in vivo along with physiological pressure and stretch. We subjected human aortic endothelial cells (HAECs) to hyperglycemic conditions under both "normal" and "disturbed" flow. Our results confirm significant and quantifiable differences in phenotypic and functional markers between cells cultured under conditions of "normal" and "disturbed flow" under hyperglycemic conditions suggesting that elevated glucose in conjunction with "disturbed" flow conditions results in significantly higher level of EC dysfunction. The ECCM can therefore be used as a physiologically relevant model to study early stage hyperglycemia induced atherosclerosis for basic research, drug discovery, and screening and toxicity studies.

Molecular insight into the effect of lipid bilayer environments on thrombospondin-1 and calreticulin interactions.

Thrombospondin-1 (TSP1) binding to cell surface calreticulin (CRT) stimulates the association of CRT with low-density lipoprotein (LDL) receptor-related protein (LRP1) to signal focal adhesion disassembly and engagement of cellular activities. A recent study demonstrated that membrane rafts are necessary for TSP1-mediated focal adhesion disassembly, but the molecular role of membrane rafts in mediating TSP1-CRT-LRP1 signaling is unknown. In this study, we investigated the effect of lipid bilayer environments on TSP1 and CRT interactions via atomically detailed molecular dynamics simulations. Results showed that the microscopic structural properties of lipid molecules and mesoscopic mechanical properties and electrostatic potential of the bilayer were significantly different between a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer and a raftlike lipid bilayer [a POPC/cholesterol (CHOL) raftlike lipid bilayer or a POPC/CHOL/sphingomyelin (SM) raftlike lipid bilayer], and the difference was enhanced by SM lipids in a raftlike lipid bilayer. These bilayer property differences affect the interactions of CRT with the bilayer, further influencing CRT conformation and TSP1-CRT interactions. A raftlike lipid bilayer stabilized CRT conformation as compared to a POPC bilayer environment. TSP1 binding to CRT resulted in a conformation for the CRT N-domain more "open" than that of the CRT P-domain in a raftlike lipid bilayer environment, which could facilitate binding of CRT to LRP1 to engage downstream signaling. The open conformational changes of CRT by binding to TSP1 in a raftlike lipid bilayer were enhanced by SM lipids in a lipid bilayer. The direct interactions of both the N- and P-domains of CRT with the bilayer contribute to the more open conformation of CRT in the TSP1-CRT complex on a raftlike lipid bilayer as compared to that on a POPC bilayer. The interactions of CRT or the TSP1-CRT complex with the lipid bilayer also caused CHOL molecules and/or lipids to be more coordinated and to aggregate into patchlike regions in the raftlike lipid bilayers. The lipid and CHOL molecule coordination and aggregation could in turn affect the interactions of CRT with the membrane raft, thereby altering TSP1-CRT interactions and CRT conformational changes that potentially regulate its interactions with LRP1. This study provides molecular insights into the role of lipid bilayer environments in TSP1-CRT interactions and in the CRT conformational changes that are predicted to facilitate binding of CRT to LRP1 to engage downstream signaling events.

Revisiting the matricellular concept.

The concept of a matricellular protein was first proposed by Paul Bornstein in the mid-1990s to account for the non-lethal phenotypes of mice with inactivated genes encoding thrombospondin-1, tenascin-C, or SPARC. It was also recognized that these extracellular matrix proteins were primarily counter or de-adhesive. This review reappraises the matricellular concept after nearly two decades of continuous investigation. The expanded matricellular family as well as the diverse and often unexpected functions, cellular location, and interacting partners/receptors of matricellular proteins are considered. Development of therapeutic strategies that target matricellular proteins are discussed in the context of pathology and regenerative medicine.

The role of matricellular proteins in glaucoma.

Glaucoma is an optic neuropathy affecting approximately 60million people worldwide and is the second most common cause of irreversible blindness. Elevated intraocular pressure (IOP) is the main risk factor for developing glaucoma and is caused by impaired aqueous humor drainage through the trabecular meshwork (TM) and Schlemm's canal (SC). In primary open angle glaucoma (POAG), this elevation in IOP in turn leads to deformation at the optic nerve head (ONH) specifically at the lamina cribrosa (LC) region where there is also a deposition of extracellular matrix (ECM) molecules such as collagen and fibronectin. Matricellular proteins are non-structural secreted glycoproteins that help cells communicate with their surrounding ECM. This family of proteins includes connective tissue growth factor (CTGF), also known as CCN2, thrombospondins (TSPs), secreted protein acidic and rich in cysteine (SPARC), periostin, osteonectin, and Tenascin-C and -X and other ECM proteins. All members appear to play a role in fibrosis and increased ECM deposition. Most are widely expressed in tissues particularly in the TM and ONH and deficiency of TSP1 and SPARC have been shown to lower IOP in mouse models of glaucoma through enhanced outflow facility. The role of these proteins in glaucoma is emerging as some have an association with the pathophysiology of the TM and LC regions and might therefore be potential targets for therapeutic intervention in glaucoma.

Calreticulin regulates transforming growth factor-ß-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). 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-ß. 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-ß stimulation of type I collagen and fibronectin. In contrast, fibroblasts with increased CRT expression had enhanced responses to TGF-ß. The lack of CRT does not impact canonical TGF-ß signaling as TGF-ß was able to stimulate Smad reporter activity in CRT-/- MEFs. CRT regulation of TGF-ß-stimulated Ca(2+) signaling is important for induction of ECM. CRT-/- MEFs failed to increase intracellular Ca(2+) levels in response to TGF-ß. NFAT activity is required for ECM stimulation by TGF-ß. In CRT-/- MEFs, TGF-ß stimulation of NFAT nuclear translocation and reporter activity is impaired. Importantly, CRT is required for TGF-ß stimulation of ECM under conditions of ER stress, as tunicamycin-induced ER stress was insufficient to induce ECM production in TGF-ß stimulated CRT-/- MEFs. Together, these data identify CRT-regulated Ca(2+)-dependent pathways as a critical molecular link between ER stress and TGF-ß fibrotic signaling.