Role for CCN1 in lysophosphatidic acid response in PC-3 human prostate cancer cells.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are bioactive phospholipids that act as mitogens in various cancers. Both LPA and S1P activate G-protein coupled receptors (GPCRs). We examined the role of CCN1/CYR61, an inducible matricellular protein, in LPA-induced signal transduction in PC-3 human prostate cancer cells. We found that both LPA and S1P induced expression of CCN1 and CCN2 within 2-4 h. CCN1 was induced by 18:1-LPA, but not by 18:0-, 18:2-, or 18:3-LPAs. A free fatty acid receptor-4 agonist inhibited LPA-induced CCN1 induction. CCN1 appeared in the ECM within 2 h after LPA addition. LPA caused biphasic activation of Erk MAPK, with an initial peak at 10-20 min followed by a later phase after 6 h. LPA increased adhesion of PC-3 cells to culture substrates (standard culture plates, fibronectin, or extracellular matrix) at 2 h, an intermediate event between early and late LPA signals. Knockdown of CCN1 suppressed LPA-induced adhesion to ECM or fibronectin. ECM from CCN1 knockdown cells was a poor substrate for adhesion, as compared to ECM from control cells. These results suggest that CCN1 contributes to LPA responses in the tumor microenvironment. The LPA-CCN1 axis holds promise for the development of novel therapeutic strategies in cancer.

The synthesis and secretion of versican isoform V3 by mammalian cells: A role for N-linked glycosylation.

Versican is a large extracellular matrix (ECM) chondroitin sulfate (CS) proteoglycan found in most soft tissues, which is encoded by the VCAN gene. At least four major isoforms (V0, V1, V2, and V3) are generated via alternative splicing. The isoforms of versican are expressed and accumulate in various tissues during development and disease, where they contribute to ECM structure, cell growth and migration, and immune regulation, among their many functions. While several studies have identified the mRNA transcript for the V3 isoform in a number of tissues, little is known about the synthesis, secretion, and targeting of the V3 protein. In this study, we used lentiviral generation of doxycycline-inducible rat V3 with a C-terminal tag in stable NIH 3T3 cell lines and demonstrated that V3 is processed through the classical secretory pathway. We further show that N-linked glycosylation is required for efficient secretion and solubility of the protein. By site-directed mutagenesis, we identified amino acids 57 and 330 as the active N-linked glycosylation sites on V3 when expressed in this cell type. Furthermore, exon deletion constructs of V3 revealed that exons 11-13, which code for portions of the carboxy region of the protein (G3 domain), are essential for V3 processing and secretion. Once secreted, the V3 protein associates with hyaluronan along the cell surface and within the surrounding ECM. These results establish critical parameters for the processing, solubility, and targeting of the V3 isoform by mammalian cells and establishes a role for V3 in the organization of hyaluronan.

The role of CCN4/WISP-1 in the cancerous phenotype.

CCN proteins are secreted into the extracellular environment where they interact with both components of the extracellular matrix and with cell surface receptors to regulate cellular function. Through these interactions, CCNs act as extracellular ligands to activate intracellular signal transduction pathways. CCN4/WISP-1, like other CCNs, plays multiple physiologic roles in development and also participates in pathogenesis. CCN4 is of particular interest with respect to cancer, showing promise as a biomarker or prognostic factor as well as a potential therapeutic target. This review focuses on recent work addressing the role of CCN4 in cancer. While CCN4 has been identified as an oncogene in a number of cancers, where it enhances cell migration and promoting epithelial-mesenchymal transition, there are other cancers where CCN4 appears to play an inhibitory role. The mechanisms underlying these differences in cellular response have not yet been delineated, but are an active area of investigation. The expression and activities of CCN4 splice variants are likewise an emerging area for study. CCN4 acts as an autocrine factor that regulates the cancer cells from which it is secreted. However, CCN4 is also a paracrine factor that is secreted by stromal fibroblasts, and can affect the function of vascular endothelial cells. In summary, current evidence is abundant in regard to establishing potential roles for CCN4 in oncogenesis, but much remains to be learned about the functions of this fascinating protein as both an autocrine and paracrine regulator in the tumor microenvironment.

Age-related accumulation of phosphorylated mitofusin 2 protein in retinal ganglion cells correlates with glaucoma progression.

Dysregulation of axonal bioenergetics is likely a key mechanism in the initiation and progression of age-related neurodegenerative diseases. Glaucoma is a quintessential neurodegenerative disorder characterized by progressive deterioration of the optic nerve (ON) and eventual death of retinal ganglion cells (RGCs). Age and elevation of intraocular pressure are key risk factors in glaucoma, but the common early hallmarks of decreased axonal transport and increased bioenergetic vulnerability likely underlie disease initiation. We examined the correlation between bioenergetics and axonal transport with mitochondrial mutation frequency and post-translational modifications of mitofusin 2 (Mfn2) in RGCs during glaucoma progression. No increase in the frequency of mtDNA mutations was detected, but we observed significant shifts in mitochondrial protein species. Mfn2 is a fusion protein that functions in mitochondrial biogenesis, maintenance, and mitochondrial transport. We demonstrate that Mfn2 accumulates selectively in RGCs during glaucomatous degeneration, that two novel states of Mfn2 exist in retina and ON, and identify a phosphorylated form that selectively accumulates in RGCs, but is absent in ON. Phosphorylation of Mfn2 is correlated with higher ubiquitination, and failure of the protein to reach the ON. Together, these data suggest that post-translational modification of Mfn2 is associated with its dysregulation during a window of metabolic vulnerability that precedes glaucomatous degeneration. Future work to either manipulate expression of Mfn2 or to prevent its degradation could have therapeutic value in the treatment of neurodegenerative diseases where long-tract axons are vulnerable.

Versican Deficiency Significantly Reduces Lung Inflammatory Response Induced by Polyinosine-Polycytidylic Acid Stimulation.

Viral infection is an exacerbating factor contributing to chronic airway diseases, such as asthma, via mechanisms that are still unclear. Polyinosine-polycytidylic acid (poly(I:C)), a Toll-like receptor 3 (TLR3) agonist used as a mimetic to study viral infection, has been shown to elicit inflammatory responses in lungs and to exacerbate pulmonary allergic reactions in animal models. Previously, we have shown that poly(I:C) stimulates lung fibroblasts to accumulate an extracellular matrix (ECM), enriched in hyaluronan (HA) and its binding partner versican, which promotes monocyte adhesion. In the current study, we aimed to determine the in vivo role of versican in mediating inflammatory responses in poly(I:C)-induced lung inflammation using a tamoxifen-inducible versican-deficient mouse model (Vcan-/- mice). In C57Bl/6 mice, poly(I:C) instillation significantly increased accumulation of versican and HA, especially in the perivascular and peribronchial regions, which were enriched in infiltrating leukocytes. In contrast, versican-deficient (Vcan-/-) lungs did not exhibit increases in versican or HA in these regions and had strikingly reduced numbers of leukocytes in the bronchoalveolar lavage fluid and lower expression of inflammatory chemokines and cytokines. Poly(I:C) stimulation of lung fibroblasts isolated from control mice generated HA-enriched cable structures in the ECM, providing a substrate for monocytic cells in vitro, whereas lung fibroblasts from Vcan-/- mice did not. Moreover, increases in proinflammatory cytokine expression were also greatly attenuated in the Vcan-/- lung fibroblasts. These findings provide strong evidence that versican is a critical inflammatory mediator during poly(I:C)-induced acute lung injury and, in association with HA, generates an ECM that promotes leukocyte infiltration and adhesion.