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.

Overexpression of SPARC in human trabecular meshwork increases intraocular pressure and alters extracellular matrix.

PURPOSE: Intraocular pressure (IOP) regulation is largely unknown. SPARC-null mice demonstrate a lower IOP resulting from increased outflow. SPARC is a matricellular protein often associated with fibrosis. We hypothesized that SPARC overexpression would alter IOP by affecting extracellular matrix (ECM) synthesis and/or turnover in the trabecular meshwork (TM). METHODS: An adenoviral vector containing human SPARC was used to increase SPARC expression in human TM endothelial cells and perfused human anterior segments using multiplicities of infection (MOIs) 25 or 50. Total RNA from TM was used for quantitative PCR, while protein from cell lysates and conditioned media were used for immunoblot analyses and zymography. After completion of perfusion, the anterior segments were fixed, sectioned, and examined by light and confocal microscopy. RESULTS: SPARC overexpression increased the IOP of perfused human anterior segments. Fibronectin and collagens IV and I protein levels were elevated in both TM cell cultures and within the juxtacanalicular (JCT) region of perfused anterior segments. Collagen VI and laminin protein levels were increased in TM cell cultures but not in perfused anterior segments. The protein levels of pro-MMP-9 decreased while the kinetic inhibitors of metalloproteinases, TIMP-1 and PAI-1 protein levels, increased at MOI 25. At MOI 50, the protein levels of pro-MMP-1, -3, and -9 also decreased while PAI-1 and TIMP-1 and -3 increased. Only MMP-9 activity was decreased on zymography. mRNA levels of the collagens, fibronectin, and laminin were not affected by SPARC overexpression. CONCLUSIONS: SPARC overexpression increases IOP in perfused cadaveric human anterior segments resulting from a qualitative change the JCT ECM. Selective decrease of MMP-9 activity is likely part of the mechanism. SPARC is a regulatory node for IOP.

SPARC fusion protein induces cellular adhesive signaling.

Secreted protein, acidic and rich in cysteine (SPARC) has been described as a counteradhesive matricellular protein with a diversity of biological functions associated with morphogenesis, remodeling, cellular migration, and proliferation. We have produced mouse SPARC with a FLAG-tag at the N-terminus of SPARC (Flag-SPARC, FSP) in a Bac-to-Bac baculoviral expression system. After affinity purification, this procedure yields SPARC of high purity, with an electrophoretic mobility of ∼44 kDa under reducing conditions, and ∼38-39 kDa under non-reducing conditions. Unexpectedly, FSP adsorbed to plastic supported cell attachment and spreading, in a calcium-dependent manner. The adhesive activity of native FSP was inhibited by prior incubation with anti-SPARC IgG. Cell adhesion to FSP induced the formation of filopodia and lamellipodia but not focal adhesions that were prominent on cells that were attached to fibronectin. In addition, FSP induced the tyrosine phosphorylation of FAK and paxillin in attached epithelial cells. Erk1/2 and Rac were also activated in cells attached to FSP, but at a lower level in comparison to cells on fibronectin. This study provides new insight into the biological functions of SPARC, a matricellular protein with important roles in cell-extracellualr matrix interactions.

Combinatorial targeting and discovery of ligand-receptors in organelles of mammalian cells.

Phage display screening allows the study of functional protein-protein interactions at the cell surface, but investigating intracellular organelles remains a challenge. Here we introduce internalizing-phage libraries to identify clones that enter mammalian cells through a receptor-independent mechanism and target-specific organelles as a tool to select ligand peptides and identify their intracellular receptors. We demonstrate that penetratin, an antennapedia-derived peptide, can be displayed on the phage envelope and mediate receptor-independent uptake of internalizing phage into cells. We also show that an internalizing-phage construct displaying an established mitochondria-specific localization signal targets mitochondria, and that an internalizing-phage random peptide library selects for peptide motifs that localize to different intracellular compartments. As a proof-of-concept, we demonstrate that one such peptide, if chemically fused to penetratin, is internalized receptor-independently, localizes to mitochondria, and promotes cell death. This combinatorial platform technology has potential applications in cell biology and drug development.

Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC.

Astrocytes regulate synaptic connectivity in the CNS through secreted signals. Here we identified two astrocyte-secreted proteins, hevin and SPARC, as regulators of excitatory synaptogenesis in vitro and in vivo. Hevin induces the formation of synapses between cultured rat retinal ganglion cells. SPARC is not synaptogenic, but specifically antagonizes synaptogenic function of hevin. Hevin and SPARC are expressed by astrocytes in the superior colliculus, the synaptic target of retinal ganglion cells, concurrent with the excitatory synaptogenesis. Hevin-null mice had fewer excitatory synapses; conversely, SPARC-null mice had increased synaptic connections in the superior colliculus. Furthermore, we found that hevin is required for the structural maturation of the retinocollicular synapses. These results identify hevin as a positive and SPARC as a negative regulator of synapse formation and signify that, through regulation of relative levels of hevin and SPARC, astrocytes might control the formation, maturation, and plasticity of synapses in vivo.

Proteolysis of the matricellular protein hevin by matrix metalloproteinase-3 produces a SPARC-like fragment (SLF) associated with neovasculature in a murine glioma model.

The matricellular SPARC-family member hevin (Sparc-like 1/SPARCL-1/SC1/Mast9) contributes to neural development and alters tumor progression in a range of mammalian models. Based on sequence similarity, we hypothesized that proteolytic digestion of hevin would result in SPARC-like fragments (SLF) that affect the activity and/or location of these proteins. Incubation of hevin with matrix metalloproteinase-3 (MMP-3), a protease known to cleave SPARC, produced a limited number of peptides. Sequencing revealed the major proteolytic products to be SPARC-like in primary structure. In gliomas implanted into murine brain, a SLF was associated with SPARC in the neovasculature but not with hevin, the latter prominent in the astrocytes encompassed by infiltrating tumor. In this model of invasive glioma that involves MMP-3 activity, host-derived SLF was not observed in the extracellular matrix adjacent to tumor cells. In contrast, it occurred with its homolog SPARC in the angiogenic response to the tumor. We conclude that MMP-3-derived SLF is a marker of neovessels in glioma, where it could influence the activity of SPARC.

Identification of a sequence in the matricellular protein SPARC that interacts with the scavenger receptor stabilin-1.

SPARC (osteonectin/BM-40), a secreted matricellular protein that promotes cellular deadhesion and motility in wound healing, carcinogenesis, and inflammation, binds to the scavenger receptor stabilin-1 in alternatively activated macrophages and undergoes endocytosis and clearance from the extracellular space. Both SPARC and stabilin-1 are expressed by endothelial cells during inflammation, but their interaction in this context is unknown. We have identified a binding site on SPARC for stabilin-1 by a solid-state peptide array coupled with a modified enzyme-linked immunosorbent assay. A monoclonal antibody that recognizes the identified binding site was also characterized that could be an inhibitor for the SPARC-stabilin-1 interaction in macrophages or endothelial cells.

Behavioral signs of chronic back pain in the SPARC-null mouse.

STUDY DESIGN: Secreted Protein, Acidic, and Rich in Cysteine (SPARC)-null mice were examined for behavioral signs of chronic low back and/or radicular pain. OBJECTIVE: to assess SPARC-null mice as an animal model of chronic low back and/or radicular pain caused by degenerative disc disease. SUMMARY OF BACKGROUND DATA: degeneration of intervertebral discs is a major cause of chronic low back and adicular pain in humans. Inactivation of the SPARC gene in mice results in premature intervertebral disc degeneration. The effect of disc degeneration on behavioral measures of chronic pain has not been evaluated in this model. METHODS: cohorts of young and old (3 and 6-12 months, respectively) SPARC-null and wild-type control mice were screened for behavioral indices of low back and/or radiating pain. Sensitivity to mechanical, cold and heat stimuli, locomotor impairment, and movement-evoked hypersensitivity were determined. Animals were challenged with 3 analgesic agents with different mechanisms: morphine, dexamethasone, and gabapentin. RESULTS: SPARC-null mice showed signs of movement-evoked discomfort as early as 3 months of age. Hypersensitivity to cold stimuli on both the lower back and hindpaws developed with increasing age. SPARC-null mice had normal sensitivity to tactile and heat stimuli, and locomotor skills were not impaired. The hypersensitivity to cold was reversed by morphine, but not by dexamethasone or gabapentin. CONCLUSION: SPARC-null mice display behavioral signs consistent with chronic low back and radicular pain that we attribute to intervertebral disc degeneration. We hypothesize that the SPARC-null mouse is useful as a model of chronic back pain due to degenerative disc disease.

Inactivation of SPARC enhances high-fat diet-induced obesity in mice.

Secreted protein, acidic and rich in cysteine (SPARC), a matricellular protein, modulates extracellular matrix assembly and turnover in many physiological processes. SPARC-null mice exhibit an increased accumulation of adipose tissue. To distinguish between the functions of SPARC in adipogenesis during development and adulthood, we studied wild-type (WT) and SPARC-null mice maintained on a normal (low-fat) or high-fat (HF) diet. On an HF diet, SPARC-null mice exhibited significantly greater weight gain, in comparison to their WT counterparts, and had an enhanced cortical bone area that was likely due to increased mechanical loading. Diet-induced obesity (DIO) was also associated with an increase in vertebral trabecular bone in WT mice, but a significant change in this parameter was not observed in SPARC-null animals. We show that SPARC inhibits mitotic clonal expansion of preadipocytes at an early stage of adipogenesis. Moreover, there were substantially diminished levels of type I collagen in SPARC-null adipose tissue, as well as a reduction in the number of cross-linked, mature collagen fibers. In the absence of SPARC, mice show enhanced DIO. In adult animals, SPARC functions in the production and remodeling of adipose tissue, as well as in the regulation of preadipocyte differentiation.

Biomaterial topography alters healing in vivo and monocyte/macrophage activation in vitro.

The effect of biomaterial topography on healing in vivo and monocyte/macrophage stimulation in vitro was assessed. A series of expanded polytetrafluoroethylene (ePTFE) materials were characterized by increasing average intranodal distance of 1.2 µm (1.2-ePTFE), 3.0 µm (3.0-ePTFE), and 4.4 µm (4.4-ePTFE), but presented consistent surface chemistry with nonporous PTFE (np-PTFE). Subcutaneous implantation of 4.4-ePTFE into mice resulted in a statistically thinner capsule that appeared less organized and less dense than the np-PTFE response. In vitro, isolated monocytes/macrophages cultured on np-PTFE produced low levels of interleukin 1-beta (IL-1ß), 1.2-ePTFE and 3.0-ePTFE stimulated intermediate levels, and 4.4-ePTFE stimulated a 15-fold increase over np-PTFE. Analysis of cDNA microarrays demonstrated that additional proinflammatory cytokines and chemokines, including IL-1ß, interleukin 6, tumor necrosis factor alpha, monocyte chemotactic protein 1, and macrophage inflammatory protein 1-beta, were expressed at higher levels by monocytes/macrophages cultured on 4.4-ePTFE at 4 and 24 h, respectively. Expression ratios for several genes were quantified by RT-PCR and were consistent with those from the cDNA array results. These results demonstrate the effect of biomaterial topography on early proinflammatory cytokine production and gene transcription by monocytes/macrophages in vitro and decreased fibrous capsule thickness in vivo.

SPARC deficiency results in improved surgical survival in a novel mouse model of glaucoma filtration surgery.

Glaucoma is a disease frequently associated with elevated intraocular pressure that can be alleviated by filtration surgery. However, the post-operative subconjunctival scarring response which blocks filtration efficiency is a major hurdle to the achievement of long-term surgical success. Current application of anti-proliferatives to modulate the scarring response is not ideal as these often give rise to sight-threatening complications. SPARC (secreted protein, acidic and rich in cysteine) is a matricellular protein involved in extracellular matrix (ECM) production and organization. In this study, we investigated post-operative surgical wound survival in an experimental glaucoma filtration model in SPARC-null mice. Loss of SPARC resulted in a marked (87.5%) surgical wound survival rate compared to 0% in wild-type (WT) counterparts. The larger SPARC-null wounds implied that aqueous filtration through the subconjunctival space was more efficient in comparison to WT wounds. The pronounced increase in both surgical survival and filtration efficiency was associated with a less collagenous ECM, smaller collagen fibril diameter, and a loosely-organized subconjunctival matrix in the SPARC-null wounds. In contrast, WT wounds exhibited a densely packed collagenous ECM with no evidence of filtration capacity. Immunolocalization assays confirmed the accumulation of ECM proteins in the WT but not in the SPARC-null wounds. The observations in vivo were corroborated by complementary data performed on WT and SPARC-null conjunctival fibroblasts in vitro. These findings indicate that depletion of SPARC bestows an inherent change in post-operative ECM remodeling to favor wound maintenance. The evidence presented in this report is strongly supportive for the targeting of SPARC to increase the success of glaucoma filtration surgery.

Processing of the matricellular protein hevin in mouse brain is dependent on ADAMTS4.

The matricellular SPARC family member hevin (SPARC-like 1/SPARCL-1/SC1/Mast9) contributes to neural development and alters tumor progression in a range of mammalian models. The distribution of hevin in mouse tissues was reexamined with a novel monoclonal antibody that discriminates between hevin and its ortholog SPARC. We now report proteolysis of hevin in many tissues, with the most extensive processing in the brain. We demonstrate a cleavage site within the hevin sequence for the neural tissue proteinase ADAMTS4. Digestion of hevin by ADAMTS4 in vitro produced fragments similar to those present in brain lysates. Monoclonal antibodies revealed a SPARC-like fragment generated from hevin that was co-localized with ADAMTS4 in vivo. We show that proteolysis of hevin by ADAMTS4 in the mouse cerebellum is important for the normal development of this tissue. In conclusion, we have identified the fragmentation of hevin by ADAMTS4 in the mouse brain and propose that this specific proteolysis is integral to cell morphology and extracellular matrix deposition in the developing brain.

Lack of host SPARC enhances vascular function and tumor spread in an orthotopic murine model of pancreatic carcinoma.

Utilizing subcutaneous tumor models, we previously validated SPARC (secreted protein acidic and rich in cysteine) as a key component of the stromal response, where it regulated tumor size, angiogenesis and extracellular matrix deposition. In the present study, we demonstrate that pancreatic tumors grown orthotopically in Sparc-null (Sparc(-/-)) mice are more metastatic than tumors grown in wild-type (Sparc(+/+)) littermates. Tumors grown in Sparc(-/-) mice display reduced deposition of fibrillar collagens I and III, basement membrane collagen IV and the collagen-associated proteoglycan decorin. In addition, microvessel density and pericyte recruitment are reduced in tumors grown in the absence of host SPARC. However, tumors from Sparc(-/-) mice display increased permeability and perfusion, and a subsequent decrease in hypoxia. Finally, we found that tumors grown in the absence of host SPARC exhibit an increase in alternatively activated macrophages. These results suggest that increased tumor burden in the absence of host SPARC is a consequence of reduced collagen deposition, a disrupted vascular basement membrane, enhanced vascular function and an immune-tolerant, pro-metastatic microenvironment.

Thrombospondins in the heart: potential functions in cardiac remodeling.

Cardiac remodeling after myocardial injury involves inflammation, angiogenesis, left ventricular hypertrophy and matrix remodeling. Thrombospondins (TSPs) belong to the group of matricellular proteins, which are non-structural extracellular matrix proteins that modulate cell-matrix interactions and cell function in injured tissues or tumors. They interact with different matrix and membrane-bound proteins due to their diverse functional domains. That the expression of TSPs strongly increases during cardiac stress or injury indicates an important role for them during cardiac remodeling. Recently, the protective properties of TSP expression against heart failure have been acknowledged. The current review will focus on the biological role of TSPs in the ischemic and hypertensive heart, and will describe the functional consequences of TSP polymorphisms in cardiac disease.

SPARC functions as an inhibitor of adipogenesis.

Adipogenesis, a key step in the pathogenesis of obesity, involves extensive ECM remodeling, changes in cell-ECM interactions, and cytoskeletal rearrangement. Matricellular proteins regulate cell-cell and cell-ECM interactions. Evidence in vivo and in vitro indicates that the prototypic matricellular protein, SPARC, inhibits adipogenesis and promotes osteoblastogenesis. Herein we discuss mechanisms underlying the inhibitory effect of SPARC on adipogenesis. SPARC enhances the Wnt/beta-catenin signaling pathway and regulates the expression and posttranslational modification of collagen. SPARC might drive preadipocytes away from the status of growth arrest and therefore prevent terminal differentiation. SPARC could also decrease WAT deposition through its negative effects on angiogenesis. Therefore, several stages of white adipose tissue accumulation are sensitive to the inhibitory effects of SPARC.

Absence of thrombospondin-2 causes age-related dilated cardiomyopathy.

BACKGROUND: The progressive shift from a young to an aged heart is characterized by alterations in the cardiac matrix. The present study investigated whether the matricellular protein thrombospondin-2 (TSP-2) may affect cardiac dimensions and function with physiological aging of the heart. METHODS AND RESULTS: TSP-2 knockout (KO) and wild-type mice were followed up to an age of 60 weeks. Survival rate, cardiac function, and morphology did not differ at a young age in TSP-2 KO compared with wild-type mice. However, >55% of the TSP-2 KO mice died between 24 and 60 weeks of age, whereas <10% of the wild-type mice died. In the absence of TSP-2, older mice displayed a severe dilated cardiomyopathy with impaired systolic function, increased cardiac dilatation, and fibrosis. Ultrastructural analysis revealed progressive myocyte stress and death, accompanied by an inflammatory response and replacement fibrosis, in aging TSP-2 KO animals, whereas capillary or coronary morphology or density was not affected. Importantly, adeno-associated virus-9 gene-mediated transfer of TSP-2 in 7-week-old TSP-2 KO mice normalized their survival and prevented dilated cardiomyopathy. In TSP-2 KO animals, age-related cardiomyopathy was accompanied by increased matrix metalloproteinase-2 and decreased tissue transglutaminase-2 activity, together with impaired collagen cross-linking. At the cardiomyocyte level, TSP-2 deficiency in vivo and its knockdown in vitro decreased the activation of the Akt survival pathway in cardiomyocytes. CONCLUSIONS: TSP-2 expression in the heart protects against age-dependent dilated cardiomyopathy.

A novel GGA-binding site is required for intracellular sorting mediated by stabilin-1.

Stabilin-1 is a unique scavenger receptor that combines endocytic and intracellular sorting functions in macrophages. Stabilin-1 mediates the endocytosis of acetylated low-density lipoprotein (acLDL), SPARC, and growth hormone family member placental lactogen (PL). At the same time, stabilin-1 is involved in trans-Golgi network-to-endosome routing of the endogenous chitinase-like protein SI-CLP (stabilin-interacting chitinase-like protein). A DDSLL motif in the cytoplasmic tail of stabilin-1 interacts with GGA adaptors; however, the deletion of DDSLL reduces but does not abrogate this interaction. Here, we identified a novel GGA-binding site, EDDADDD, in the cytoplasmic tail of stabilin-1. The deletion of EDDADDD impaired and the deletion of both the DDSLL and EDDADDD sites abrogated the interaction of stabilin-1 with GGAs. The surface exposure of stabilin-1 and stabilin-1-mediated endocytosis of acLDL, SPARC, and PL were not affected by the deletion either of DDSLL or EDDADDD or both. At the same time, both GGA-binding sites were necessary for the intracellular sorting of SI-CLP performed by stabilin-1. Our data indicate that the novel GGA-binding site EDDADDD is essential for stabilin-1-mediated intracellular sorting but is not required for endocytosis.

Extracellular and intracellular mechanisms that mediate the metastatic activity of exogenous osteopontin.

BACKGROUND: Osteopontin affects several steps of the metastatic cascade. Despite direct correlation with metastasis in experimental systems and in patient studies, the extracellular and intracellular basis for these observations remains unsolved. In this study, the authors used human melanoma and sarcoma cell lines to evaluate the effects of soluble osteopontin on metastasis. METHODS: Exogenous osteopontin or negative controls, including a site-directed mutant osteopontin, were used in functional assays in vitro, ex vivo, and in vivo that were designed to test the extracellular and intracellular mechanisms involved in experimental metastasis. RESULTS: In the extracellular environment, the results confirmed that soluble osteopontin is required for its prometastatic effects; this phenomenon is specific, arginine-glycine-aspartic acid (RGD)-dependent, and evident in experimental models of metastasis. In the intracellular environment, osteopontin initially induced rapid tyrosine 418 (Tyr-418) dephosphorylation of the cellular homolog of the Rous sarcoma virus (c-Src), with decreases in actin stress fibers and increased binding to the vascular endothelium. This heretofore undescribed Tyr dephosphorylation was followed by a tandem c-Src phosphorylation after tumor cell attachment to the metastatic site. CONCLUSIONS: The results of this study revealed a complex molecular interaction as well as a dual role for osteopontin in metastasis that depends on whether tumor cells are in circulation or attached. Such context-dependent functional insights may contribute to antimetastasis strategies.

SPARC-null mice exhibit lower intraocular pressures.

PURPOSE: SPARC is a matricellular protein that is highly expressed in remodeling tissues, including the trabecular meshwork and ciliary body. The hypothesis for the study was that SPARC contributes to the regulation of intraocular pressure (IOP). The IOPs of SPARC-null mice, their corresponding wild-type (WT), and heterozygous animals were compared. METHODS: Diurnal and nocturnal IOPs of C57Bl/6x129SvJ WT, SPARC-null, and heterozygous mice were measured. Fluorophotometric measurements were made to assess aqueous turnover. Central corneal thickness (CCT) was measured using histology, ultrasound biomicroscopy, and optical coherence tomography. Iridocorneal angles were examined using light microscopy (LM). RESULTS: During the day, the mean IOP of SPARC-null mice (n = 142, 16.9 +/- 2.4 mm Hg) was lower than that of both WT mice (n = 104, 19.9 +/- 2.9 mm Hg; P < 10(-12)), and heterozygotes (n = 38, 19.3 +/- 2.5 mm Hg; P < 10(-4)). At night, SPARC-null mice also exhibited a blunted increase in IOP in comparison to WT and heterozygous mice. CCTs were not significantly different between WT and SPARC-null mice. Heterozygous mice tended to have thicker corneas (3.4%). Fluorophotometric measurements suggest that aqueous turnover rates in SPARC-null mice are equal to if not greater than rates in WT mice. LM of the SPARC-null iridocorneal angle revealed morphology that is indistinguishable from WT. CONCLUSIONS: SPARC-null mice have lower IOPs than do their WT counterparts with equal CCTs. The rate of aqueous turnover suggests that the mechanism is enhanced outflow resistance.

SPARC inhibits adipogenesis by its enhancement of beta-catenin signaling.

SPARC (secreted protein acidic and rich in cysteine) modulates interactions between cells and extracellular matrix and is enriched in white adipose tissue. We have reported that SPARC-null mice accumulate significantly more fat than wild-type mice and maintain relatively high levels of serum leptin. We now show that SPARC inhibits adipogenesis in vitro. Specifically, recombinant SPARC inhibited (a) adipocyte differentiation of stromal-vascular cells isolated from murine white adipose tissue and (b) the expression of adipogenic transcription factors and adipocyte-specific genes. SPARC induced the accumulation and nuclear translocation of beta-catenin and subsequently enhanced the interaction of beta-catenin and T cell/lymphoid enhancer factor 1. The activity of integrin-linked kinase was required for the effect of SPARC on beta-catenin accumulation as well as extracellular matrix remodeling. During adipogenesis, fusiform preadipocytes change into sphere-shaped adipocytes and convert the extracellular matrix from a fibronectin-rich stroma to a laminin-rich basal lamina. SPARC retarded the morphological changes exhibited by preadipocytes during differentiation. In the presence of SPARC, the deposition of fibronectin was enhanced, and that of laminin was inhibited; in parallel, the expression of alpha5 integrin was enhanced, and that of alpha6 integrin was inhibited. Lithium chloride, which enhances the accumulation of beta-catenin, also inhibited the expression of alpha6 integrin. These findings demonstrate a role for SPARC in adipocyte morphogenesis and in signaling processes leading to terminal differentiation.