Secreted Protein Acidic and Rich in Cysteine Is a Matrix Scavenger Chaperone

Secreted Protein Acidic and Rich in Cysteine (SPARC) is one of the major non-structural proteins of the extracellular matrix (ECM) in remodeling tissues. The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. Although SPARC has been shown to act as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture, no plausible molecular mechanism of its action has been proposed. In the present study, we demonstrate that SPARC mediates the disassembly and degradation of ECM networks by functioning as a matricellular chaperone. While it has low affinity to its targets inside the cells where the Ca2+ concentrations are low, high extracellular concentrations of Ca2+ activate binding to multiple ECM proteins, including collagens. We demonstrated that in vitro, this leads to the inhibition of collagen I fibrillogenesis and disassembly of pre-formed collagen I fibrils by SPARC at high Ca2+ concentrations. In cell culture, exogenous SPARC was internalized by the fibroblast cells in a time- and concentration-dependent manner. Pulse-chase assay further revealed that internalized SPARC is quickly released outside the cell, demonstrating that SPARC shuttles between the cell and ECM. Fluorescently labeled collagen I, fibronectin, vitronectin, and laminin were co-internalized with SPARC by fibroblasts, and semi-quantitative Western blot showed that SPARC mediates internalization of collagen I. Using a novel 3-dimentional model of fluorescent ECM networks pre-deposited by live fibroblasts, we demonstrated that degradation of ECM depends on the chaperone activity of SPARC. These results indicate that SPARC may represent a new class of scavenger chaperones, which mediate ECM degradation, remodeling and repair by disassembling ECM networks and shuttling ECM proteins into the cell. Further understanding of this mechanism may provide insight into the pathogenesis of matrix-associated disorders and lead to the novel treatment strategies

Apigenin Inhibits UVB-Induced Skin Carcinogenesis: The Role of Thrombospondin-1 as an Anti-Inflammatory Factor

We have previously demonstrated that apigenin promotes the expression of antiangiogenic protein thrombospondin-1 (TSP1) via a mechanism driven by mRNA-binding protein HuR. Here, we generated a novel mouse model with whole-body THBS-1 gene knockout on SKH-1 genetic background, which allows studies of UVB-induced acute skin damage and carcinogenesis and tests TSP1 involvement in apigenin's anticancer effects. Apigenin significantly inhibited UVB-induced carcinogenesis in the wild-type (WT) animals but not in TSP1 KO (TKO) mice, suggesting that TSP1 is a critical component of apigenin's chemopreventive function in UVB-induced skin cancer. Importantly, TKO mice presented with the elevated cutaneous inflammation at baseline, which was manifested by increased inflammatory infiltrates (neutrophils and macrophages) and elevated levels of the two key inflammatory cytokines, IL-6 and IL-12. In agreement, maintaining normal TSP1 expression in the UVB-irradiated skin of WT mice using topical apigenin application caused a marked decrease of circulating inflammatory cytokines. Finally, TKO mice showed an altered population dynamics of the bone marrow myeloid progenitor cells (CD11b+), with dramatic expansion of the population of neutrophil progenitors (Ly6ClowLy6Ghigh) compared to the WT control. Our results indicate that the cutaneous tumor suppressor TSP1 is a critical mediator of the in vivo anticancer effect of apigenin in skin, specifically of its anti-inflammatory action

Single administration of p2TA (AB103), a CD28 antagonist peptide, prevents inflammatory and thrombotic reactions and protects against gastrointestinal injury in total-body irradiated mice.

The goal of this study was to elucidate the action of the CD28 mimetic peptide p2TA (AB103) that attenuates an excessive inflammatory response in mitigating radiation-induced inflammatory injuries. BALB/c and A/J mice were divided into four groups: Control (C), Peptide (P; 5 mg/kg of p2TA peptide), Radiation (R; total body irradiation with 8 Gy γ-rays), and Radiation + Peptide (RP; irradiation followed by p2TA peptide 24 h later). Gastrointestinal tissue damage was evaluated by analysis of jejunum histopathology and immunohistochemistry for cell proliferation (Cyclin D1) and inflammation (COX-2) markers, as well as the presence of macrophages (F4/80). Pro-inflammatory cytokines IL-6 and KC as well as fibrinogen were quantified in plasma samples obtained from the same mice. Our results demonstrated that administration of p2TA peptide significantly reduced the irradiation-induced increase of IL-6 and fibrinogen in plasma 7 days after exposure. Seven days after total body irradiation with 8 Gy of gamma rays numbers of intestinal crypt cells were reduced and villi were shorter in irradiated animals compared to the controls. The p2TA peptide delivery 24 h after irradiation led to improved morphology of villi and crypts, increased Cyclin D1 expression, decreased COX-2 staining and decreased numbers of macrophages in small intestine of irradiated mice. Our study suggests that attenuation of CD28 signaling is a promising therapeutic approach for mitigation of radiation-induced tissue injury

Immunohistochemistry of activated macrophage marker F4/80 in mouse jejunum.

<p>Jejunal tissues were collected 7 days after irradiation and embedded in paraffin. After fixation and processing, cross sections of jejunum were immunostained with F4/80 antibodies and number of F4/80 positive cells per mm² area of tissue was counted using HistoQuest image analysis software. Bars represent the means ± SEMs for tissue sections of mice in each group. *Statistically significant differences between R and RP groups (P<0.05).</p

The effect of p<i>2TA</i> peptide on COX-2 expression in jejunal crypts.

<p>Expression of COX-2 in mouse intestine: a–h) overview, i–p) details. a–d, i–l) A/J mice; e–h, m–p) BALB/c mice; a,e,i,m) sham irradiated controls; b,f,j,n) mice treated with p<i>2TA</i> peptide six days before tissue harvest; c,g,k,o) mice exposed to 8 Gy gamma rays total body irradiation seven days before the sacrifice; d,h,l,p) mice exposed to 8 Gy gamma rays that received p<i>2TA</i> peptide 24 hours after radiation exposure. In irradiated mice (c,g,k,o) villus integrity is disrupted, their crypts show disorganization, and COX-2 staining is observable in cells in the surface layer of villi. Intestines of irradiated mice that were also treated with 5 mg/kg p<i>2TA</i> peptide 24 hours after irradiation show a more normal morphology with respect to villus height and crypt appearance, and they also show fewer COX-2 positive cells in villi surface cell layer, while COX-2 staining pattern inside villi replicates the pattern seen in sham irradiated mice.</p

The effect of p<i>2TA</i> peptide on jejunal crypts.

<p>Jejunal tissues were collected 7 days after irradiation and embedded in paraffin. After fixation and processing, cross sections of jejunum were stained with hematoxylin and eosin and analyzed for the number of surviving crypts (panel <b>A</b>) and villus height (panel <b>B</b>). Bars represent the means ± SEM of 4 cross-sections for each mouse, and 4–5 mice in each group. *Statistically significant difference between R and RP groups (P<0.05).</p

The Chemopreventive Bioflavonoid Apigenin Inhibits Prostate Cancer Cell Motility through the Focal Adhesion Kinase/Src Signaling Mechanism

Prostate cancer mortality is primarily attributed to metastatic rather than primary, organ-confined disease. Acquiring a motile and invasive phenotype is an important step in development of tumors and ultimately metastasis. This step involves remodeling of the extracellular matrix and of cell-matrix interactions, cell movement mediated by the actin cytoskeleton, and activation of focal adhesion kinase (FAK)/Src signaling. Epidemiologic studies suggest that the metastatic behavior of prostate cancer may be an ideal target for chemoprevention. The natural flavone apigenin is known to have chemopreventive properties against many cancers, including prostate cancer. Here, we study the effect of apigenin on motility, invasion, and its mechanism of action in metastatic prostate carcinoma cells (PC3-M). We found that apigenin inhibits PC3-M cell motility in a scratch-wound assay. Live cell imaging studies show that apigenin diminishes the speed and affects directionality of cell motion. Alterations in the cytoskeleton are consistent with impaired cell movement in apigenin-treated cells. Apigenin treatment leads to formation of &quot;exaggerated filopodia,&quot; which show accumulation of focal adhesion proteins at their tips. Furthermore, apigenin-treated cells adhere more strongly to the extracellular matrix. Additionally, apigenin decreases activation of FAK and Src, and phosphorylation of Src substrates FAK Y576/577 and Y925. Expression of constitutively active Src blunts the effect of apigenin on cell motility and cytoskeleton remodeling. These results show that apigenin inhibits motility and invasion of prostate carcinoma cells, disrupts actin cytoskeleton organization, and inhibits FAK/Src signaling. These studies provide mechanistic insight into developing novel strategies for inhibiting prostate cancer cell motility and invasiveness

The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

<div><p>Plakoglobin (PG) is an armadillo protein that associates with both classic and desmosomal cadherins, but is primarily concentrated in mature desmosomes in epithelia. While reduced levels of PG have been reported in localized and hormone refractory prostate tumors, the functional significance of these changes is unknown. Here we report that PG expression is reduced in samples of a prostate tumor tissue array and inversely correlated with advancing tumor potential in 7 PCa cell lines. Ectopically expressed PG enhanced intercellular adhesive strength, and attenuated the motility and invasion of aggressive cell lines, whereas silencing PG in less tumorigenic cells had the opposite effect. PG also regulated cell-substrate adhesion and motility through extracellular matrix (ECM)-dependent inhibition of Src kinase, suggesting that PG’s effects were not due solely to increased intercellular adhesion. PG silencing resulted in elevated levels of the ECM protein vitronectin (VN), and exposing PG-expressing cells to VN induced Src activity. Furthermore, increased VN levels and Src activation correlated with diminished expression of PG in patient tissues. Thus, PG may inhibit Src by keeping VN low. Our results suggest that loss of intercellular adhesion due to reduced PG expression might be exacerbated by activation of Src through a PG-dependent mechanism. Furthermore, PG down-regulation during PCa progression could contribute to the known VN-dependent promotion of PCa invasion and metastasis, demonstrating a novel functional interaction between desmosomal cell-cell adhesion and cell-substrate adhesion signaling axes in prostate cancer.</p> </div