Secreted protein acidic and rich in cysteine (SPARC) knockout mice have greater outflow facility.

PURPOSE: Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that regulates intraocular pressure (IOP) by altering extracellular matrix (ECM) homeostasis within the trabecular meshwork (TM). We hypothesized that the lower IOP previously observed in SPARC -/- mice is due to a greater outflow facility. METHODS: Mouse outflow facility (Clive) was determined by multiple flow rate infusion, and episcleral venous pressure (Pe) was estimated by manometry. The animals were then euthanized, eliminating aqueous formation rate (Fin) and Pe. The C value was determined again (Cdead) while Fin was reduced to zero. Additional mice were euthanized for immunohistochemistry to analyze ECM components of the TM. RESULTS: The Clive and Cdead of SPARC -/- mice were 0.014 ± 0.002 µL/min/mmHg and 0.015 ± 0.002 µL/min/mmHg, respectively (p = 0.376, N/S). Compared to the Clive = 0.010 ± 0.002 µL/min/mmHg and Cdead = 0.011 ± 0.002 µL/min/mmHg in the WT mice (p = 0.548, N/S), the Clive and Cdead values for the SPARC -/- mice were higher. Pe values were estimated to be 8.0 ± 0.2 mmHg and 8.3 ± 0.7 mmHg in SPARC -/- and WT mice, respectively (p = 0.304, N/S). Uveoscleral outflow (Fu) was 0.019 ± 0.007 µL/min and 0.022 ± 0.006 µL/min for SPARC -/- and WT mice, respectively (p = 0.561, N/S). Fin was 0.114 ± 0.002 µL/min and 0.120 ± 0.016 µL/min for SPARC -/- and WT mice (p = 0.591, N/S). Immunohistochemistry demonstrated decreases of collagen types IV and VI, fibronectin, laminin, PAI-1, and tenascin-C within the TM of SPARC -/- mice (p < 0.05). CONCLUSIONS: The lower IOP of SPARC -/- mice is due to greater aqueous humor outflow facility through the conventional pathway. Corresponding changes in several matricellular proteins and ECM structural components were noted in the TM of SPARC -/- mice.

The predicted collagen-binding domains of Drosophila SPARC are essential for survival and for collagen IV distribution and assembly into basement membranes.

The assembly of basement membranes (BMs) into tissue-specific morphoregulatory structures requires non-core BM components. Work in Drosophila indicates a principal role of collagen-binding matricellular glycoprotein SPARC (Secreted Protein, Acidic, Rich in Cysteine) in larval fat body BM assembly. We report that SPARC and collagen IV (Col(IV)) first colocalize in the trans-Golgi of hemocyte-like cell lines. Mutating the collagen-binding domains of Drosophila SPARC led to the loss of colocalization with Col(IV), a fibrotic-like BM, and 2nd instar larval lethality, indicating that SPARC binding to Col(IV) is essential for survival. Analysis of this mutant at 2nd instar reveals increased Col(IV) puncta within adipocytes, reflecting a disruption in the intracellular chaperone-like activity of SPARC. Removal of the disulfide bridge in the C-terminal EF-hand2 of SPARC, which is known to enhance Col(IV) binding, did not lead to larval lethality; however, a less intense fat body phenotype was observed. Additionally, both SPARC mutants exhibited altered fat body BM pore topography. Wing imaginal disc-derived SPARC did not localize within Col(IV)-rich matrices. This raises the possibility that SPARC interaction with Col(IV) requires initial intracellular interaction to colocalize at the BM or that wing-derived SPARC undergoes differential post-translational modifications that impacts its function. Collectively, these data provide evidence that the chaperone-like activity of SPARC on Col(IV) begins just prior to their co-secretion and demonstrate for the first time that the Col(IV) chaperone-like activity of SPARC is necessary for Drosophila development beyond the 2nd instar.

Interleukin-8 as a therapeutic target for chronic low back pain: Upregulation in human cerebrospinal fluid and pre-clinical validation with chronic reparixin in the SPARC-null mouse model.

BACKGROUND: Low back pain (LBP) is the leading global cause of disability and is associated with intervertebral disc degeneration (DD) in some individuals. However, many adults have DD without LBP. Understanding why DD is painful in some and not others may unmask novel therapies for chronic LBP. The objectives of this study were to a) identify factors in human cerebrospinal fluid (CSF) associated with chronic LBP and b) examine their therapeutic utility in a proof-of-concept pre-clinical study. METHODS: Pain-free human subjects without DD, pain-free human subjects with DD, and patients with chronic LBP linked to DD were recruited and lumbar MRIs, pain and disability levels were obtained. CSF was collected and analyzed by multiplex cytokine assay. Interleukin-8 (IL-8) expression was confirmed by ELISA in CSF and in intervertebral discs. The SPARC-null mouse model of progressive, age-dependent DD and chronic LBP was used for pre-clinical validation. Male SPARC-null and control mice received systemic Reparixin, a CXCR1/2 (receptors for IL-8 and murine analogues) inhibitor, for 8 weeks. Behavioral signs of axial discomfort and radiating pain were assessed. Following completion of the study, discs were excised and cultured, and conditioned media was evaluated with a protein array. FINDINGS: IL-8 was elevated in CSF of chronic LBP patients with DD compared to pain-free subjects with or without DD. Chronic inhibition with reparixin alleviated low back pain behaviors and attenuated disc inflammation in SPARC-null mice. INTERPRETATION: These studies suggest that the IL-8 signaling pathway is a viable therapy for chronic LBP. FUND: Supported by NIH, MMF, CIHR and FRQS.

SPARC is required for the maintenance of glucose homeostasis and insulin secretion in mice.

Obesity, metabolic syndrome, and type 2 diabetes, three strongly interrelated diseases, are associated to increased morbidity and mortality worldwide. The pathogenesis of obesity-associated disorders is still under study. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein expressed in many cell types including adipocytes, parenchymal, and non-parenchymal hepatic cells and pancreatic cells. Studies have demonstrated that SPARC inhibits adipogenesis and promotes insulin resistance; in addition, circulating SPARC levels were positively correlated with body mass index in obese individuals. Therefore, SPARC is being proposed as a key factor in the pathogenesis of obesity-associated disorders. The aim of this study is to elucidate the role of SPARC in glucose homeostasis. We show here that SPARC null (SPARC-/-) mice displayed an abnormal insulin-regulated glucose metabolism. SPARC-/- mice presented an increased adipose tissue deposition and an impaired glucose homeostasis as animals aged. In addition, the absence of SPARC worsens high-fat diet-induced diabetes in mice. Interestingly, although SPARC-/- mice on high-fat diet were sensitive to insulin they showed an impaired insulin secretion capacity. Of note, the expression of glucose transporter 2 in islets of SPARC-/- mice was dramatically reduced. The present study provides the first evidence that deleted SPARC expression causes diabetes in mice. Thus, SPARC deficient mice constitute a valuable model for studies concerning obesity and its related metabolic complications, including diabetes.

Dysregulation of the Inflammatory Mediators in the Multifidus Muscle After Spontaneous Intervertebral Disc Degeneration SPARC-null Mice is Ameliorated by Physical Activity.

STUDY DESIGN: A longitudinal case-control animal model. OBJECTIVE: The aim of this study was to investigate the inflammatory pathways active in the multifidus muscle after spontaneous intervertebral disc degeneration (IDD), and whether these IDD-related muscle changes can be ameliorated by exercise. SUMMARY OF BACKGROUND DATA: A pro-inflammatory response is present in the multifidus muscle after an intervertebral disc lesion and has been proposed to drive the structural alterations present during low back pain. However, it is not known whether spontaneous IDD produces an inflammatory response. Furthermore, exercise/physical activity produces a strong anti-inflammatory response, but its effectiveness in ameliorating inflammation in the multifidus is unknown. We assessed the inflammatory profile of the multifidus and the effectiveness of physical activity as a treatment using an animal model of spontaneous model of IDD. METHODS: Wild-type and SPARC null mice that were sedentary or housed with a running wheel were used in this study. Multifidus muscle segments were harvested from L2-L6 from the mice at 9 months of age after they had undergone a magnetic resonance imaging (MRI) scan to determine levels with IDD. The inflammatory profile of the multifidus was examined using quantitative polymerase chain reaction (PCR) assays. RESULTS: Spontaneous IDD in the SPARC-null mice caused a dysregulation of interleukin (IL)-1ß, IL6, transforming growth factor-beta (TGFß1), and adiponectin expression. More specifically, the proximity and degree of IDD was related to levels of IL-1ß expression. Physical activity reduced the pro-inflammatory response to IDD in the multifidus. IL-1ß, tumor necrosis factor (TNF), IL-10, adiponectin, and leptin levels were lower in the physically active group. CONCLUSION: These results reveal that spontaneous IDD causes dysregulation of the inflammatory pathways active in the multifidus muscle. These alterations were related to the severity of IDD and were prevented by physical activity. LEVEL OF EVIDENCE: N/A.

Pleiotropic roles of the matricellular protein Sparc in tendon maturation and ageing.

Acute and chronic tendinopathies remain clinically challenging and tendons are predisposed to degeneration or injury with age. Despite the high prevalence of tendon disease in the elderly, our current understanding of the mechanisms underlying the age-dependent deterioration of tendon function remains very limited. Here, we show that Secreted protein acidic and rich in cysteine (Sparc) expression significantly decreases in healthy-aged mouse Achilles tendons. Loss of Sparc results in tendon collagen fibrillogenesis defects and Sparc-/- tendons are less able to withstand force in comparison with their respective wild type counterparts. On the cellular level, Sparc-null and healthy-aged tendon-derived cells exhibited a more contracted phenotype and an altered actin cytoskeleton. Additionally, an elevated expression of the adipogenic marker genes PPARγ and Cebpα with a concomitant increase in lipid deposits in aged and Sparc-/- tendons was observed. In summary, we propose that Sparc levels in tendons are critical for proper collagen fibril maturation and its age-related decrease, together with a change in ECM properties favors lipid accretion in tendons.

Mesenchymal Transition of High-Grade Breast Carcinomas Depends on Extracellular Matrix Control of Myeloid Suppressor Cell Activity.

The extracellular matrix (ECM) contributes to the biological and clinical heterogeneity of breast cancer, and different prognostic groups can be identified according to specific ECM signatures. In high-grade, but not low-grade, tumors, an ECM signature characterized by high SPARC expression (ECM3) identifies tumors with increased epithelial-to-mesenchymal transition (EMT), reduced treatment response, and poor prognosis. To better understand how this ECM3 signature is contributing to tumorigenesis, we expressed SPARC in isogenic cell lines and found that SPARC overexpression in tumor cells reduces their growth rate and induces EMT. SPARC expression also results in the formation of a highly immunosuppressive microenvironment, composed by infiltrating T regulatory cells, mast cells, and myeloid-derived suppressor cells (MDSCs). The ability of SPARC to induce EMT depended on the localization and suppressive function of myeloid cells, and inhibition of the suppressive function MDSCs by administration of aminobisphosphonates could revert EMT, rendering SPARC-overexpressing tumor cells sensitive to Doxil. We conclude that that SPARC is regulating the interplay between MDSCs and the ECM to drive the induction of EMT in tumor cells.

Healthy and tumoral tissue resistivity in wild-type and sparc-/- animal models.

Despite the technological improvement of radiologic, endoscopic and nuclear imaging, the accuracy of diagnostic procedures for tumors can be limited whenever a mass-forming lesion is identified. This is true also because bioptical sampling cannot be properly guided into the lesions so as to puncture neoplastic tissue and to avoid necrotic areas. Under these circumstances, invasive and expensive procedures are still required to obtain diagnosis which is mandatory to plan the most appropriate therapeutic strategy. In order to test if electrical impedance spectroscopy may be helpful in providing further evidence for cancer detection, resistivity measurements were taken on 22 mice, 11 wild-type and 11 sparc-/- (knock out for the protein SPARC: secreted protein acidic and rich in cysteine), bearing mammary carcinomas, by placing a needle-probe into tumor, peritumoral and contralateral healthy fat areas. Tumor resistivity was significantly lower than both peritumoral fat and contralateral fat tissues. Resistivity in sparc-/- mice was lower than wild-type animals. A significant frequency dependence of resistivity was present in tissues analyzed. We conclude that accurate measurements of resistivity may allow to discriminate between tissues with different pathological and/or structural characteristics. Therefore, resistivity measurements could be considered for in vivo detection and differential diagnosis of tumor masses.

Decreased Mechanical Strength and Collagen Content in SPARC-Null Periodontal Ligament Is Reversed by Inhibition of Transglutaminase Activity.

The periodontal ligament (PDL) is a critical tissue that provides a physical link between the mineralized outer layer of the tooth and the alveolar bone. The PDL is composed primarily of nonmineralized fibrillar collagens. Expression of secreted protein acidic and rich in cysteine (SPARC/osteonectin), a collagen-binding matricellular protein, has been shown to be essential for collagen homeostasis in PDL. In the absence of SPARC, PDL collagen fibers are smaller and less dense than fibers that constitute WT PDL. The aim of this study was to identify cellular mechanisms by which SPARC affected collagen fiber assembly and morphology in PDL. Cross-linking of fibrillar collagens is one parameter that is known to affect insoluble collagen incorporation and fiber morphology. Herein, the reduction in collagen fiber size and quantity in the absence of SPARC expression was shown to result in a PDL with reduced molar extraction force in comparison to that of WT mice (C57Bl/6J). Furthermore, an increase in transglutaminase activity was found in SPARC-null PDL by biochemical analyses that was supported by immunohistochemical results. Specifically, collagen I was identified as a substrate for transglutaminase in PDL and transglutaminase activity on collagen I was found to be greater in SPARC-null tissues in comparison to WT. Strikingly, inhibition of transglutaminase activity in SPARC-null PDL resulted in increases in both collagen fiber thickness and in collagen content, whereas transglutaminase inhibitors injected into WT mice resulted in increases in collagen fiber thickness only. Furthermore, PDL treated with transglutaminase inhibitors exhibited increases in molar extraction force in WT and in SPARC-null mice. Thus, SPARC is proposed to act as a critical regulator of transglutaminase activity on collagen I with implications for mechanical strength of tissues.

Secreted protein acidic and rich in cysteine facilitates age-related cardiac inflammation and macrophage M1 polarization.

To investigate the role of secreted protein acidic and rich in cysteine (SPARC) in age-related cardiac inflammation, we studied six groups of mice: young (3-5 mo old), middle-aged (10-12 mo old), and old (18-29 mo old) C57BL/6 wild-type (WT) and SPARC-null (Null) mice (n = 7-10/group). Cardiac function and structure were determined by echocardiography. The left ventricle was used for cytokine gene array and macrophage quantification by immunohistochemistry. Macrophage infiltration increased with age in WT (n = 5-6/group, P < 0.05 for young vs. old), but not in Null. Proinflammatory markers (Ccl5, Cx3cl1, Ccr2, and Cxcr3) increased in middle-aged and old WT, whereas they were increased only in old Null compared with respective young (n = 5-6/group, P < 0.05 for all). These results suggest that SPARC deletion delayed age-related cardiac inflammation. To further assess how SPARC affects inflammation, we stimulated peritoneal macrophages with SPARC (n = 4). SPARC treatment increased expression of proinflammatory macrophage M1 markers and decreased anti-inflammatory M2 markers. Echocardiography (n = 7-10/group) revealed an age-related increase in wall thickness of the left ventricle in WT (0.76 ± 0.02 mm in young vs. 0.91 ± 0.03 mm in old; P < 0.05) but not in Null (0.78 ± 0.01 mm in young vs. 0.84 ± 0.02 mm in old). In conclusion, SPARC deletion delayed age-related increases in macrophage infiltration and proinflammatory cytokine expression in vivo and in vitro. SPARC acts as an important mediator of age-related cardiac inflammation by increasing the expression of macrophage M1 markers and decreasing M2 markers.

Loss of Sparc in p53-null Astrocytes Promotes Macrophage Activation and Phagocytosis Resulting in Decreased Tumor Size and Tumor Cell Survival.

Both the induction of SPARC expression and the loss of the p53 tumor suppressor gene are changes that occur early in glioma development. Both SPARC and p53 regulate glioma cell survival by inverse effects on apoptotic signaling. Therefore, during glioma formation, the upregulation of SPARC may cooperate with the loss of p53 to enhance cell survival. This study determined whether the loss of Sparc in astrocytes that are null for p53 would result in reduced cell survival and tumor formation and increased tumor immunogenicity in an in vivo xenograft brain tumor model. In vitro, the loss of Sparc in p53-null astrocytes resulted in an increase in cell proliferation, but a loss of tumorigenicity. At 7 days after intracranial implantation, Sparc-null tumors had decreased tumor cell survival, proliferation and reduced tumor size. The loss of Sparc promoted microglia/macrophage activation and phagocytosis of tumor cells. Our results indicate that the loss of p53 by deletion/mutation in the early stages of glioma formation may cooperate with the induction of SPARC to potentiate cancer cell survival and escape from immune surveillance.

Loss of SPARC in mouse skeletal muscle causes myofiber atrophy.

INTRODUCTION: The expression of secreted protein acidic and rich in cysteine (SPARC) in skeletal muscle decreases with age. Here, we examined the role of SPARC in skeletal muscle by reducing its expression. METHODS: SPARC expression was suppressed by introducing short interfering RNA (siRNA) into mouse tibialis anterior muscle. Myofiber diameter, atrogin1, and muscle RING-finger protein 1 (MuRF1) expression, and tumor necrosis factor-α (TNFα) and transforming growth factor-ß (TGFß) signaling were then analyzed. RESULTS: Reduced SPARC expression caused decreases in the diameter of myofibers, especially fast-type ones, accompanied by upregulation of atrogin1, but not MuRF1, at 10 days after siRNA transfection. The expression of TNFα and TGFß and the phosphorylation status of p38 were not affected by SPARC knockdown, whereas Smad3 phosphorylation was increased at 2 days after siRNA transfection. CONCLUSIONS: The loss of SPARC not only upregulates atrogin1 expression but also enhances TGFß signaling, which may in turn cause muscle atrophy.

Patterns of gene expression in microarrays and expressed sequence tags from normal and cataractous lenses.

In this contribution, we have examined the patterns of gene expression in normal and cataractous lenses as presented in five different papers using microarrays and expressed sequence tags. The purpose was to evaluate unique and common patterns of gene expression during development, aging and cataracts.

Stromal SPARC contributes to the detrimental fibrotic changes associated with myeloproliferation whereas its deficiency favors myeloid cell expansion.

In myeloid malignancies, the neoplastic clone outgrows normal hematopoietic cells toward BM failure. This event is also sustained by detrimental stromal changes, such as BM fibrosis and osteosclerosis, whose occurrence is harbinger of a dismal prognosis. We show that the matricellular protein SPARC contributes to the BM stromal response to myeloproliferation. The degree of SPARC expression in BM stromal elements, including CD146(+) mesenchymal stromal cells, correlates with the degree of stromal changes, and the severity of BM failure characterizing the prototypical myeloproliferative neoplasm primary myelofibrosis. Using Sparc(-/-) mice and BM chimeras, we demonstrate that SPARC contributes to the development of significant stromal fibrosis in a model of thrombopoietin-induced myelofibrosis. We found that SPARC deficiency in the radioresistant BM stroma compartment impairs myelofibrosis but, at the same time, associates with an enhanced reactive myeloproliferative response to thrombopoietin. The link betwen SPARC stromal deficiency and enhanced myeloid cell expansion under a myeloproliferative spur is also supported by the myeloproliferative phenotype resulting from the transplantation of defective Apc(min) mutant hematopoietic cells into Sparc(-/-) but not WT recipient BM stroma. Our results highlight a complex influence of SPARC over the stromal and hematopoietic BM response in myeloproliferative conditions.

Lumbar intervertebral disc degeneration associated with axial and radiating low back pain in ageing SPARC-null mice.

Chronic low back pain (LBP) is a complex, multifactorial disorder with unclear underlying mechanisms. In humans and rodents, decreased expression of secreted protein acidic rich in cysteine (SPARC) is associated with intervertebral disc (IVD) degeneration and signs of LBP. The current study investigates the hypothesis that IVD degeneration is a risk factor for chronic LBP. SPARC-null and age-matched control mice ranging from 6 to 78 weeks of age were evaluated in this study. X-ray and histologic analysis revealed reduced IVD height, increased wedging, and signs of degeneration (bulging and herniation). Cutaneous sensitivity to cold, heat, and mechanical stimuli were used as measures of referred (low back and tail) and radiating pain (hind paw). Region specificity was assessed by measuring icilin- and capsaicin-evoked behaviour after subcutaneous injection into the hind paw or upper lip. Axial discomfort was measured by the tail suspension and grip force assays. Motor impairment was determined by the accelerating rotarod. Physical function was evaluated by voluntary activity after axial strain or during ambulation with forced lateral flexion. SPARC-null mice developed (1) region-specific, age-dependent hypersensitivity to cold, icilin, and capsaicin (hind paw only), (2) axial discomfort, (3) motor impairment, and (4) reduced physical function. Morphine (6 mg/kg, i.p.) reduced cutaneous sensitivity and alleviated axial discomfort in SPARC-null mice. Ageing SPARC-null mice mirror many aspects of the complex and challenging nature of LBP in humans and incorporate both anatomic and functional components of the disease. The current study supports the hypothesis that IVD degeneration is a risk factor for chronic LBP.

Losartan slows pancreatic tumor progression and extends survival of SPARC-null mice by abrogating aberrant TGFß activation.

Pancreatic adenocarcinoma, a desmoplastic disease, is the fourth leading cause of cancer-related death in the Western world due, in large part, to locally invasive primary tumor growth and ensuing metastasis. SPARC is a matricellular protein that governs extracellular matrix (ECM) deposition and maturation during tissue remodeling, particularly, during wound healing and tumorigenesis. In the present study, we sought to determine the mechanism by which lack of host SPARC alters the tumor microenvironment and enhances invasion and metastasis of an orthotopic model of pancreatic cancer. We identified that levels of active TGFß1 were increased significantly in tumors grown in SPARC-null mice. TGFß1 contributes to many aspects of tumor development including metastasis, endothelial cell permeability, inflammation and fibrosis, all of which are altered in the absence of stromal-derived SPARC. Given these results, we performed a survival study to assess the contribution of increased TGFß1 activity to tumor progression in SPARC-null mice using losartan, an angiotensin II type 1 receptor antagonist that diminishes TGFß1 expression and activation in vivo. Tumors grown in SPARC-null mice progressed more quickly than those grown in wild-type littermates leading to a significant reduction in median survival. However, median survival of SPARC-null animals treated with losartan was extended to that of losartan-treated wild-type controls. In addition, losartan abrogated TGFß induced gene expression, reduced local invasion and metastasis, decreased vascular permeability and altered the immune profile of tumors grown in SPARC-null mice. These data support the concept that aberrant TGFß1-activation in the absence of host SPARC contributes significantly to tumor progression and suggests that SPARC, by controlling ECM deposition and maturation, can regulate TGFß availability and activation.

SPARC-like 1 (SC1) is a diversely expressed and developmentally regulated matricellular protein that does not compensate for the absence of SPARC in the CNS.

SPARC-like 1 (SC1) is a member of the SPARC family of matricellular proteins that has been implicated in the regulation of processes such as cell migration, proliferation, and differentiation. Here we show that SC1 exhibits remarkably diverse and dynamic expression in the developing and adult nervous system. During development, SC1 localizes to radial glia and pial-derived structures, including the vasculature, choroid plexus, and pial membranes. SC1 is not downregulated in postnatal development, but its expression shifts to distinct time windows in subtypes of glia and neurons, including astrocytes, large projection neurons, Bergmann glia, Schwann cells, and ganglionic satellite cells. In addition, SC1 expression levels and patterns are not altered in the SPARC null mouse, suggesting that SC1 does not compensate for the absence of SPARC. We conclude that SC1 and SPARC may share significant homology, but are likely to have distinct but complementary roles in nervous system development.

Deletion of the SPARC acidic domain or EGF-like module reduces SPARC-induced migration and signaling through p38 MAPK/HSP27 in glioma.

We previously demonstrated that secreted protein acidic and rich in cysteine (SPARC) increases heat shock protein 27 (HSP27) expression and phosphorylation and promotes glioma cell migration through the p38 mitogen-activated protein kinase (MAPK)/HSP27 signaling pathway. As different regions of the SPARC protein mediate different SPARC functions, elucidating which SPARC domains regulate HSP27 expression, signaling and migration might provide potential therapeutic strategies to target these functions. To investigate the roles of specific domains, we used an SPARC-green fluorescent protein (GFP) fusion protein and constructs of SPARC-GFP with deletions of either the acidic domain (ΔAcidic) or the epidermal growth factor (EGF)-like module (ΔEGF). GFP, SPARC-GFP and the two deletion mutants were expressed in U87MG glioma cells. Characterization of the derived stable clones by confocal imaging and western blotting suggests proper folding, processing and secretion of the deletion constructs. Uptake of the constructs by naive cells suggests enhanced internalization of ΔAcidic and reduced internalization of ΔEGF. Wound and transwell migration assays and western blot analysis confirm our previous results and indicate that ΔAcidic reduces SPARC-induced migration and p38 MAPK/HSP27 signaling and ΔEGF decreases SPARC-induced migration and dramatically decreases the expression and phosphorylation of HSP27 but is poorly internalized. Loss of the EGF-like module suppresses the enhanced HSP27 protein stability conferred by SPARC. In conclusion, deletions of the acidic domain and EGF-like module have differential effects on cell surface binding and HSP27 protein stability; however, both regions regulate SPARC-induced migration and signaling through HSP27. Our data link the domains of SPARC with different functions and suggest one or both of the constructs as potential therapeutic agents to inhibit SPARC-induced migration.

The impact of the ovarian microenvironment on the anti-tumor effect of SPARC on ovarian cancer.

A lack of host-derived SPARC promotes disease progression in an intraperitoneal (IP) ID8 mouse model of epithelial ovarian cancer (EOC). Since orthotopic injection (OT) of ID8 cells better recapitulates high-grade serous cancer, we examined the impact of host-derived SPARC following OT injection. Sparc(-/-) and wild-type (WT) mice were injected with ID8 cells either OT or IP and tumors were analyzed at the moribund stage. Sparc(-/-) mice had reduced survival and fewer well-defined abdominal lesions compared with WT controls after IP injection, whereas no differences were observed in survival or abdominal lesions between Sparc(-/-) and WT mice after OT injection. No differences in mass or collagen content were observed in ovarian tumors between OT-injected Sparc(-/-) and WT mice. The abdominal wall of the IP-injected Sparc(-/-) mice exhibited immature and less abundant collagen fibrils compared with WT mice both in injected and non-injected controls. In contrast to human EOC, SPARC was expressed by the tumor cells but was absent in reactive stroma of WT mice. Exposure to the ovarian microenvironment through OT injections alters the metastatic behaviour of ID8 cells, which is not affected by the absence of host-derived SPARC.