Mechanical Tension in Syndecan-1 is Regulated by Extracellular Mechanical Cues and Fluidic Shear Stress

The endothelium plays a central role in regulating vascular homeostasis and is key in determining the response to materials implanted in the vascular system. Endothelial cells are uniquely sensitive to biophysical cues from applied forces and their local cellular microenvironment. The glycocalyx is a layer of proteoglycans, glycoproteins and glycosaminoglycans that lines the luminal surface of the vascular endothelium, interacting directly with the components of the blood and the forces of blood flow. In this work, we examined the changes in mechanical tension of syndecan-1, a cell surface proteoglycan that is an integral part of the glycocalyx, in response to substrate stiffness and fluidic shear stress. Our studies demonstrate that syndecan-1 is mechanically responsive to extracellular mechanical cues and alters its association with cytoskeletal and adhesion-related proteins in response to substrate stiffness and physiological flow.

Heparan Sulfate Proteoglycans in Human Colorectal Cancer

Colorectal cancer is the third most common cancer worldwide, accounting for more than 610,000 mortalities every year. Prognosis of patients is highly dependent on the disease stage at diagnosis. Therefore, it is crucial to investigate molecules involved in colorectal cancer tumorigenesis, with possible use as tumor markers. Heparan sulfate proteoglycans are complex molecules present in the cell membrane and extracellular matrix, which play vital roles in cell adhesion, migration, proliferation, and signaling pathways. In colorectal cancer, the cell surface proteoglycan syndecan-2 is upregulated and increases cell migration. Moreover, expression of syndecan-1 and syndecan-4, generally antitumor molecules, is reduced. Levels of glypicans and perlecan are also altered in colorectal cancer; however, their role in tumor progression is not fully understood. In addition, studies have reported increased heparan sulfate remodeling enzymes, as the endosulfatases. Therefore, heparan sulfate proteoglycans are candidate molecules to clarify colorectal cancer tumorigenesis, as well as important targets to therapy and diagnosis.

N-sulfation of heparan sulfate is critical for syndecan-4-mediated podocyte cell-matrix interactions

Previous research has shown that podocytes unable to assemble heparan sulfate on cell surface proteoglycan core proteins have compromised cell-matrix interactions. This report further explores the role of N-sulfation of intact heparan chains in podocyte-matrix interactions. For the purposes of this study, a murine model in which the enzyme N-deacetylase/ N-sulfotransferase 1 (NDST1) was specifically deleted in podocytes and immortalized podocyte cell lines lacking NDST1 were developed and used to explore the effects of such a mutation on podocyte behavior in vitro. NDST1 is a bifunctional enzyme, ultimately responsible for N-sulfation of heparan glycosaminoglycans produced by cells. Immunostaining of glomeruli from mice whose podocytes were null for Ndst1 ( Ndst1−/−) showed a disrupted pattern of localization for the cell surface proteoglycan, syndecan-4, and for α-actinin-4 compared with controls. The pattern of immunostaining for synaptopodin and nephrin did not show as significant alterations. In vitro studies showed that Ndst1−/− podocytes attached, spread, and migrated less efficiently than Ndst1+/+ podocytes. Immunostaining in vitro for several markers for molecules involved in cell-matrix interactions showed that Ndst1−/− cells had decreased clustering of syndecan-4 and decreased recruitment of protein kinase-Cα, α-actinin-4, vinculin, and phospho-focal adhesion kinase to focal adhesions. Total intracellular phospho-focal adhesion kinase was decreased in Ndst1−/− compared with Ndst1+/+ cells. A significant decrease in the abundance of activated integrin α5β1 on the cell surface of Ndst1−/− cells compared with Ndst1+/+ cells was observed. These results serve to highlight the critical role of heparan sulfate N-sulfation in facilitating normal podocyte-matrix interactions.

Abstract 125: The E2F1 Transcription Factor Suppresses Cardiac Fibrosis via Downregulating Syndecan-4 Expression and Smad2/3 Phosphorylation

Rationale: The E2F1 transcription factor is best known as a cell-cycle regulator; recent reports suggest its important role in cardiovascular system. Objective: To determine whether E2F1 regulates cardiac fibrosis. Methods and Results: Following Angiotensin II (Ang II) administration, E2F1-null (E2F1-KO) mice displayed a more severe cardiac fibrosis and higher levels of cardiac phospho-Smad2 (pSmad2) and pSmad3 than WT mice. Consistently, levels of pSmad2 and pSmad3 were elevated in E2F1-KO fibroblasts, which was associated with a significantly increased expression of collagen I (Col I) and α-smooth muscle actin (α-SMA) and interestingly, cell surface proteoglycan syndecan-4 (Sdc4). Knockdown of Sdc4 significantly attenuated the elevation in Smad2/3 phosphorylation and Col I and α-SMA expression in E2F1-KO fibroblasts. Remarkably, either chemical inhibition of Smad2/3 signaling or morpholino-mediated knockdown of Sdc4 expression abrogated the difference in the degree of Ang II-induced cardiac fibrosis between WT and E2F1-KO animals. Conclusions: E2F1 suppresses Ang II-induced cardiac fibrosis by downregulating Sdc4 expression and Sdc4-mediated Smad 2/3 activation.

Integrin-specific signaling pathways controlling focal adhesion formation and cell migration

The fibronectin (FN)-binding integrins α4β1 and α5β1 confer different cell adhesive properties, particularly with respect to focal adhesion formation and migration. After analyses of α4+/α5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with α4β1 and α5β1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties. First, α5β1 and α4β1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; α5β1 requires a proteoglycan coreceptor (syndecan-4), and α4β1 does not. Second, adhesion via α5β1 caused an eightfold increase in protein kinase Cα (PKCα) activation, but only basal PKCα activity was observed after adhesion via α4β1. Pharmacological inhibition of PKCα and transient expression of dominant-negative PKCα, but not dominant-negative PKCδ or PKCζ constructs, suppressed focal adhesion formation and cell migration mediated by α5β1, but had no effect on α4β1. These findings demonstrate that different integrins can signal to induce focal adhesion formation and migration by different mechanisms, and they identify PKCα signaling as central to the functional differences between α4β1 and α5β1.