TRPM8 is required for survival and radioresistance of glioblastoma cells.

TRPM8 is a Ca2+-permeable nonselective cation channel belonging to the melastatin sub-group of the transient receptor potential (TRP) family. TRPM8 is aberrantly overexpressed in a variety of tumor entities including glioblastoma multiforme where it reportedly contributes to tumor invasion. The present study aimed to disclose further functions of TRPM8 in glioma biology in particular upon cell injury by ionizing radiation. To this end, TCGA data base was queried to expose the TRPM8 mRNA abundance in human glioblastoma specimens and immunoblotting was performed to analyze the TRPM8 protein abundance in primary cultures of human glioblastoma. Moreover, human glioblastoma cell lines were irradiated with 6 MV photons and TRPM8 channels were targeted pharmacologically or by RNA interference. TRPM8 abundance, Ca2+ signaling and resulting K+ channel activity, chemotaxis, cell migration, clonogenic survival, DNA repair, apoptotic cell death, and cell cycle control were determined by qRT-PCR, fura-2 Ca2+ imaging, patch-clamp recording, transfilter migration assay, wound healing assay, colony formation assay, immunohistology, flow cytometry, and immunoblotting. As a result, human glioblastoma upregulates TRPM8 channels to variable extent. TRPM8 inhibition or knockdown slowed down cell migration and chemotaxis, attenuated DNA repair and clonogenic survival, triggered apoptotic cell death, impaired cell cycle and radiosensitized glioblastoma cells. Mechanistically, ionizing radiation activated and upregulated TRPM8-mediated Ca2+ signaling that interfered with cell cycle control probably via CaMKII, cdc25C and cdc2. Combined, our data suggest that TRPM8 channels contribute to spreading, survival and radioresistance of human glioblastoma and, therefore, might represent a promising target in future anti-glioblastoma therapy.

Actomyosin contractility and RhoGTPases affect cell-polarity and directional migration during haptotaxis.

Although much is known about chemotaxis- induced by gradients of soluble chemical cues - the molecular mechanisms involved in haptotaxis (migration induced by substrate-bound protein gradients) are largely unknown. We used micropatterning to produce discontinuous gradients consisting of µm-sized fibronectin-dots arranged at constant lateral but continuously decreasing axial spacing. Parameters like gradient slope, protein concentration and size or shape of the fibronectin dots were modified to determine optimal conditions for directional cell migration in gradient patterns. We demonstrate that fibroblasts predominantly migrate uphill towards a higher fibronectin density in gradients with a dot size of 2 × 2 µm, a 2% and 6% slope, and a low fibronectin concentration of 1 µg ml-1. Increasing dot size to 3.5 × 3.5 µm resulted in stationary cells, whereas rectangular dots (2 × 3 µm) orientated perpendicular to the gradient axis preferentially induce lateral migration. During haptotaxis, the Golgi apparatus reorients to a posterior position between the nucleus and the trailing edge. Using pharmacological inhibitors, we demonstrate that actomyosin contractility and microtubule dynamics are a prerequisite for gradient recognition indicating that asymmetric intracellular forces are necessary to read the axis of adhesive gradients. In the haptotaxis signalling cascade, RhoA and Cdc42, and the atypical protein kinase C zeta (aPKCζ), but not Rac, are located upstream of actomyosin contractility.

Subcellular stretch-induced cytoskeletal response of single fibroblasts within 3D designer scaffolds.

In vivo, cells are exposed to mechanical forces in many different ways. These forces can strongly influence cell functions or may even lead to diseases. Through their sensing machinery, cells are able to perceive the physical information of the extracellular matrix and translate it into biochemical signals resulting in cellular responses. Here, by virtue of two-component polymer scaffolds made via direct laser writing, we precisely control the cell matrix adhesions regarding their spatial arrangement and size. This leads to highly controlled and uniform cell morphologies, thereby allowing for averaging over the results obtained from several different individual cells, enabling quantitative analysis. We transiently deform these elastic structures by a micromanipulator, which exerts controlled stretching forces on primary fibroblasts grown in these scaffolds on a subcellular level. We find stretch-induced remodeling of both actin cytoskeleton and cell matrix adhesions. The responses to static and periodic stretching are significantly different. The amount of paxillin and phosphorylated focal adhesion kinase increases in cell matrix adhesions at the manipulated pillar after static stretching whereas it decreases after periodic stretching.