Absence Of Secreted Protein Acidic And Rich In Cysteine Research Articles

The Influence of the Extracellular Matrix in Inflammation: Findings from the SPARC-Null Mouse.

Matricellular proteins are secreted proteins that, among other functions, can contribute to extracellular matrix (ECM) assembly including modulation of cell:ECM interactions. Recent discoveries have indicated a fundamental role for the ECM in the regulation of inflammatory responses including cell extravasation and recruitment, immune cell differentiation, polarization, activation, and retention in tissues. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular collagen-binding protein implicated in fibrillar collagen assembly in the ECM of connective tissue as well as in basal lamina organization. Functions of SPARC in modulating cell adhesion events are also reported. Studies of phenotypic responses observed in SPARC-null mice to a variety of injury models have yielded interesting insight into the functional importance of SPARC production and aberrations in ECM structure that occur in the absence of SPARC that influence immune cell behavior and inflammatory pathways. In this review, we will discuss several examples from different tissues in which SPARC-null mice exhibited an inflammatory response distinct from those of SPARC expressing mice and provide insight into novel ECM-dependent mechanisms that influence these responses. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.

Downregulation of SPARC Is Associated with Epithelial-Mesenchymal Transition and Low Differentiation State of Biliary Tract Cancer Cells

Background: Biliary tract cancers (BTCs) have a poor prognosis. BTCs are characterized by a prominent desmoplastic reaction which possibly contributes to the aggressive phenotype of this tumor. The desmoplastic reaction includes excessive production and deposition of extracellular matrix proteins such as periostin, secreted protein acidic and rich in cysteine (SPARC), thrombospondin-1, as well as accumulation of α-smooth muscle actin-positive cancer-associated fibroblasts and immune cells, secreting growth factors and cytokines including transforming growth factor (TGF)-β. In the present study, we investigated the expression of SPARC in BTC as well as its possible regulation by TGF-β. Methods: Expression levels of Sparc, TGF-β1 and its receptor ALK5 were evaluated by quantitative real-time PCR in 6 biliary tract cell lines as well as 1 immortalized cholangiocyte cell line (MMNK-1). RNAs from tumor samples of 7 biliary tract cancer patients were analyzed for expression of Sparc, TGF-β type II receptor (TbRII) as well as Twist and ZO-1. MMNK-1 cells were stimulated with TGF-β for 24 h, and Sparc, ZO-1 and E-Cadherin expressions were determined. The presence of SPARC protein was analyzed by immunohistochemistry in tumor specimens from 10 patients. Results: When comparing basal Sparc transcript levels in diverse BTC cell lines to MMNK-1 cells, we found that it was strongly downregulated in all cancer cell lines. The remaining expression levels were higher in highly differentiated cell lines (CCSW1, MZChA1, MZChA2 and TFK-1) than in less differentiated and undifferentiated ones (BDC, SKChA1). Expression of Sparc in BTC patient samples showed a significant positive correlation with expression of the epithelial marker ZO-1. In contrast, the mesenchymal marker Twist and the TbRII showed a trend of negative correlation with expression of Sparc in these samples. TGF-β exposure significantly downregulated Sparc expression in MMNK-1 cholangiocytes in vitro in parallel to downregulation of epithelial markers (E-Cadherin and ZO-1). Finally, SPARC immunostaining was performed in 10 patient samples, and the correlation between absence of SPARC and survival times was analyzed. Conclusions: These data imply that a decrease in SPARC expression is correlated with dedifferentiation of BTC cells resulting in enhanced EMT being possibly mediated by TGF-β. Thereby SPARC levels might be a marker for individual prognosis of a patient, and strategies aiming at inhibition of SPARC downregulation might have potential for new future therapies.

Reduced fibrillar collagen accumulation in skeletal muscle of secreted protein acidic and rich in cysteine (SPARC)-null mice.

We have previously shown that secreted protein acidic and rich in cysteine (SPARC) promotes myogenic differentiation of rat skeletal muscle progenitor cells in vitro, and in vivo small interfering RNA (siRNA)-mediated transient suppression of SPARC expression in skeletal muscle of mice causes atrophic changes of myofibers, suggesting that SPARC plays a role in the maintenance of skeletal muscle function. In order to know the effect of long-term deficiency of SPARC on skeletal muscle, we performed phenotypic analyses of skeletal muscle of SPARC-null mice. Age-associated changes of myofiber diameters were comparable between wild type (WT) and SPARC-null mice at all ages examined, indicating that the growth of myofibers is unaffected by the absence of SPARC. On the other hand, accumulation of fibrillar collagen was significantly reduced in SPARC-null mice compared to WT mice after 5 months of age without significant changes of collagen I gene expression. The results obtained in the present study suggest that SPARC plays a role to maintain the stiffness of skeletal muscle by regulating collagen accumulation.

Age and SPARC dependent cardiac collagen changes (1120.7)

Secreted protein acidic and rich in cysteine (SPARC), a collagen‐binding matricellular protein, has been implicated in procollagen processing. The aim of this study was to investigate age and SPARC dependent changes in cardiac extracellular matrix (ECM). We studied 6 groups of mice (n=4/group): young (4‐5 month old), middle‐aged (11‐12 m.o.), and old (18‐23 m.o.) C57BL/6J wild type and SPARC‐null mice. The left ventricle was decellularized in 1% SDS with protease inhibitors in PBS to enrich for ECM proteins. Protein extracts (10 μg) were separated by SDS‐PAGE, digested in‐gel and analyzed by HPLC‐ESI‐MS/MS. Relative quantification was by spectral counting (Scaffold). We identified 321 proteins, of which 27 proteins were ECM or ECM‐related proteins. Of these proteins, 16 showed age‐dependent changes. Collagens I, III, IV and VI, dystrophin, and laminin b2 increased with age, which may explain previously reported age‐associated increases in cardiac stiffness. SPARC deletion blunted the age changes observed in collagens I and III and laminin b2. Of interest, fibrillin and elastin showed a significant increase with age in the Null mice, suggestive of increased microfibril deposition in the absence of SPARC. In summary, our data suggest SPARC is a possible therapeutic target for aging induced cardiac dysfunction.Grant Funding Source: Supported by HHSN 268201000036C (N01‐HV‐00244), R01 HL075360, HL051971, 1SC2 HL101430, 5I01BX000505

Age and SPARC change the extracellular matrix composition of the left ventricle.

Secreted protein acidic and rich in cysteine (SPARC), a collagen-binding matricellular protein, has been implicated in procollagen processing and deposition. The aim of this study was to investigate age- and SPARC-dependent changes in protein composition of the cardiac extracellular matrix (ECM). We studied 6 groups of mice (n = 4/group): young (4-5 months old), middle-aged (11-12 m.o.), and old (18–29 m.o.) C57BL/6J wild type (WT) and SPARC null. The left ventricle (LV) was decellularized to enrich for ECM proteins. Protein extracts were separated by SDS-PAGE, digested in-gel, and analyzed by HPLC-ESI-MS/MS. Relative quantification was performed by spectral counting, and changes in specific proteins were validated by immunoblotting. We identified 321 proteins, of which 44 proteins were extracellular proteins. Of these proteins, collagen III levels were lower in the old null mice compared to WT, suggestive of a role for SPARC in collagen deposition. Additionally, fibrillin showed a significant increase in the null middle-aged group, suggestive of increased microfibril deposition in the absence of SPARC. Collagen VI increased with age in both genotypes (>3-fold), while collagen IV showed increased age-associated levels only in the WT animals (4-fold, P < 0.05). These changes may explain the previously reported age-associated increases in LV stiffness. In summary, our data suggest SPARC is a possible therapeutic target for aging induced LV dysfunction.

SPARC regulates collagen interaction with cardiac fibroblast cell surfaces

Cardiac tissue from mice that do not express secreted protein acidic and rich in cysteine (SPARC) have reduced amounts of insoluble collagen content at baseline and in response to pressure overload hypertrophy compared with wild-type (WT) mice. However, the cellular mechanism by which SPARC affects myocardial collagen is not clearly defined. Although expression of SPARC by cardiac myocytes has been detected in vitro, immunohistochemistry of hearts demonstrated SPARC staining primarily associated with interstitial fibroblastic cells. Primary cardiac fibroblasts isolated from SPARC-null and WT mice were assayed for collagen I synthesis by [(3)H]proline incorporation into procollagen and by immunoblot analysis of procollagen processing. Bacterial collagenase was used to discern intracellular from extracellular forms of collagen I. Increased amounts of collagen I were found associated with SPARC-null versus WT cells, and the proportion of total collagen I detected on SPARC-null fibroblasts without propeptides [collagen-α(1)(I)] was higher than in WT cells. In addition, the amount of total collagen sensitive to collagenase digestion (extracellular) was greater in SPARC-null cells than in WT cells, indicating an increase in cell surface-associated collagen in the absence of SPARC. Furthermore, higher levels of collagen type V, a fibrillar collagen implicated in collagen fibril initiation, were found in SPARC-null fibroblasts. The absence of SPARC did not result in significant differences in proliferation or in decreased production of procollagen I by cardiac fibroblasts. We conclude that SPARC regulates collagen in the heart by modulating procollagen processing and interactions with fibroblast cell surfaces. These results are consistent with decreased levels of interstitial collagen in the hearts of SPARC-null mice being due primarily to inefficient collagen deposition into the extracellular matrix rather than to differences in collagen production.

SPARC mediates early extracellular matrix remodeling following myocardial infarction

Secreted protein, acidic, and rich in cysteine (SPARC) is a matricellular protein that functions in the extracellular processing of newly synthesized collagen. Collagen deposition to form a scar is a key event following a myocardial infarction (MI). Because the roles of SPARC in the early post-MI setting have not been defined, we examined age-matched wild-type (WT; n=22) and SPARC-deficient (null; n=25) mice at day 3 post-MI. Day 0 WT (n=28) and null (n=20) mice served as controls. Infarct size was 52 ± 2% for WT and 47 ± 2% for SPARC null (P=NS), indicating that the MI injury was comparable in the two groups. By echocardiography, WT mice increased end-diastolic volumes from 45 ± 2 to 83 ± 5 μl (P < 0.05). SPARC null mice also increased end-diastolic volumes but to a lesser extent than WT (39 ± 3 to 63 ± 5 μl; P < 0.05 vs. day 0 controls and vs. WT day 3 MI). Ejection fraction fell post-MI in WT mice from 57 ± 2 to 19 ± 1%. The decrease in ejection fraction was attenuated in the absence of SPARC (65 ± 2 to 28 ± 2%). Fibroblasts isolated from SPARC null left ventricle (LV) showed differences in the expression of 22 genes encoding extracellular matrix and adhesion molecule genes, including fibronectin, connective tissue growth factor (CTGF; CCN2), matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of metalloproteinase-2 (TIMP-2). The change in fibroblast gene expression levels was mirrored in tissue protein extracts for fibronectin, CTGF, and MMP-3 but not TIMP-2. Combined, the results of this study indicate that SPARC deletion preserves LV function at day 3 post-MI but may be detrimental for the long-term response due to impaired fibroblast activation.

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.

Regulates Early Fibroblast Responses to Myocardial Infarction

Secreted protein, acidic, and rich in cysteine (SPARC) is a matricellular protein that helps collagen fibrils assemble. Collagen deposition to form a scar is a key event following a myocardial infarction (MI). Accordingly, we examined wild type (WT; n=22) and SPARC deficient (SPARC‐null; n=25) mice at day 3 post‐MI. Day 0 unoperated WT (n=28) and SPARC‐null (n=20) mice served as controls. Infarct size was similar between the two groups (52±2% for WT and 47±2% for SPARC‐null; p=n.s.). WT mice increased end diastolic volumes, but SPARC‐null mice showed increased end diastolic volumes to a lesser extent, indicating reduced remodeling. Interestingly, collagen levels in the SPARC‐null mice were 66% of the wild type values, indicating reduced collagen deposition in the absence of SPARC. Fibroblasts isolated from the infarct region of the null mice showed differential expression of several ECM genes, including MMP‐3, TIMP‐2, CTGF, and TGFβ. Our data suggests that SPARC deletion is beneficial in the early time post MI by regulating fibroblast activation, but whether this benefit persists through longer times needs to be examined. In conclusion, SPARC plays a major role in cardiac wound healing by regulating fibroblast function.

SPARC Inhibits Adipogenesis by Its Enhancement of β-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.

Development of UV-induced squamous cell carcinomas is suppressed in the absence of SPARC.

SPARC (Secreted Protein Acidic and Rich in Cysteine) is a multifunctional glycoprotein belonging to a group of matrix-associated factors that mediate cell-extracellular matrix interactions but have no structural roles. In the present study we investigated the contribution of SPARC to factors that influence the development of skin tumors in response to UV irradiation. A hairless SPARC-null mouse was developed and compared to control SKH1 hairless mice in terms of skin tumor induction and extracellular matrix changes occurring in response to UV-irradiation. Following 23 weeks of exposure to UVB totaling 14.5 J per cm(2), tumor development in the wild-type mice was severe, with an average of over 20 tumors per mouse, many of which were squamous cell carcinomas. Conversely, the SPARC-null mice were strikingly tumor-resistant, developing no squamous cell carcinomas and averaging less than one small papilloma per mouse. SPARC was undetectable immunohistochemically in skin from the non-irradiated control group yet was present in relatively high quantities in the basal and superficial areas of the tumor mass. The SPARC-null mice also exhibited a limited contact hypersensitivity response and were refractory to UV induced immune suppression. In conclusion, SPARC appears to have a crucial role in mediating tumor formation in response to UV irradiation.

Alterations in the lens capsule contribute to cataractogenesis in SPARC-null mice.

The lens capsule, which is also called the lens basement membrane, is a specialized extracellular matrix produced anteriorly by the lens epithelium and posteriorly by newly differentiated fiber cells. SPARC (secreted protein, acidic and rich in cysteine) is a matricellular glycoprotein that regulates cell-cell and cell-matrix interactions, cellular proliferation and differentiation, and the expression of genes encoding extracellular matrix components. SPARC-null mice exhibit lens opacity 1 month after birth and mature cataract and capsular rupture at 5-7 months. In this study, we report disruption of the structural integrity of the lens capsule in mice lacking SPARC. The major structural protein of basement membrane, collagen type IV, in the lens capsule was substantially altered in the absence of SPARC. The lens cells immediately beneath the capsule showed aberrant morphology, with numerous protrusions into the lens basement membrane. SPARC-null lenses at 1 month of age exhibited an increased penetration of dye or radioactive tracer through the capsule, as well as a higher content of water than their wild-type counterparts. Moreover, SPARC-null fibers exhibited swelling as early as 1 month of age; by 3 months, all the fiber cells appeared swollen to a marked degree. By contrast, the absence of SPARC had no apparent morphological effect on the early stages of lens formation, cell proliferation or fiber cell differentiation. Degradation of crystallins and MIP 26, or changes in the levels of these proteins, were not detected. These results underscore the importance of the capsular extracellular matrix in the maintenance of lens transparency and indicate that SPARC participates in the synthesis, assembly and/or stabilization of the lens basement membrane.

Lenses of SPARC-null Mice Exhibit an Abnormal Cell Surface–Basement Membrane Interface

SPARC (secreted protein acidic and rich in cysteine) is a matricellular protein involved in cell–matrix interactions. We have shown previously that mice deficient in SPARC develop posterior cortical cataract early in life that progresses to a mature opacity and capsule rupture. To evaluate the primary effects of SPARC deficiency in the lens, we examined the lenses of SPARC-null and wild-type mice by electron microscopy and immunohistochemistry to investigate whether ultrastructural abnormalities occur at the basement membrane (capsule)-lens cell interface in SPARC-null mice. The most notable feature in the lenses of SPARC-null mice, relative to wild-type animals, was the modification of the basal surface of the lens epithelial and fiber cells at the basement membrane (capsule) interface. Electron microscopy revealed numerous filopodial projections of the basal surface of the lens epithelial and fiber cells into the extracellular matrix of the anterior, posterior, and equatorial regions of the lens capsule. In 1 week old precataractous lenses, basal invasive filopodia projecting into the capsule were small and infrequent. Both the size and frequency of these filopodia increased in precataractous 3–4 week old lenses and were prominent in the cataractous 5–6 week old lenses. By rhodamine-phalloidin labeling, we confirmed the presence of basal invasive filopodia projecting into the lens capsule and demonstrated that the projections contained actin filaments. In contrast to the obvious abnormal projections at the interface between the basal surface of the lens epithelial and fiber cells and the lens capsule, the apical and lateral plasma membranes of lens epithelial cells and lens fibers in SPARC-null mice were as smooth as those of wild-type mice. We conclude that the absence of SPARC in the murine lens is associated with a filopodial protrusion of the basal surface of the lens epithelium and differentiating fiber cells into the lens capsule. The altered structures appear prior to the opacification of the lens in the SPARC-null model. These observations are consistent with one or more functions previously proposed for SPARC as a modulator of cell shape and cell–matrix interactions.

Collagen accumulation is decreased in SPARC-null mice with bleomycin-induced pulmonary fibrosis.

Secreted protein acidic and rich in cysteine (SPARC) has been shown to be coexpressed with type I collagen in tissues undergoing remodeling and wound repair. We speculated that SPARC is required for the accumulation of collagen in lung injury and that its absence would attenuate collagen accumulation. Accordingly, we have assessed levels of collagen in SPARC-null mice in an intratracheal bleomycin-injury model of pulmonary fibrosis. Eight- to ten-week-old SPARC-null and wild-type (WT) mice received bleomycin (0.0035 U/g) or saline intratracheally and were subsequently killed after 14 days. Relative levels of SPARC mRNA were increased 2.7-fold (P < 0.001) in bleomycin-treated WT lungs in comparison with saline-treated lungs. Protein from bleomycin-treated WT lung contained significantly more hydroxyproline (191.9 microg/lung) than protein from either bleomycin-treated SPARC-null lungs or saline-treated WT and SPARC-null lungs (147.4 microg/lung, 125.4 microg/lung, and 113. 0 microg/lung, respectively; P < 0.03). These results indicate that SPARC is increased in response to lung injury and that accumulation of collagen, as indicated by hydroxyproline content, is attenuated in the absence of SPARC. The properties of SPARC as a matricellular protein associated with cell proliferation and matrix turnover are consistent with its participation in the development of pulmonary fibrosis.