Differential Expression of Splicing Variants of the Human Caldesmon Gene (CALD1) in Glioma Neovascularization versus Normal Brain Microvasculature

Abstract

Caldesmon is a cytoskeleton-associated protein whichhas not yet been related to neoplastic angiogenesis. In this study we investigated the expression of the caldesmon gene (CALD1) splicing variants and the protein expression level in glioma microvessels versus normal brain microvasculature. To exclude sources of splice variant expression from non-vascular components all possible cellular components present in control and glioma samples were prescreened by laser-capture microdissection followed by RT-PCR before the cohort study. We discovered differential expression of the splicing variants of CALD1 in the tumor microvessels in contrast to normal brain microvasculature. Missplicing of exons 1, 1 ؉ 4, and 1 ؉ 4 of the gene is exclusively found in glioma microvessels. To exclude the possibility that this missplicing results from splice-site mutations, Genome-wide analyses have revealed that 40 to 60% of human genes undergo alternative splicing. 1 Alternative splicing, therefore, seems to contribute considerably to enable the highly complex and diverse functions encoded by the human genome. Alternative splicing permits vertebrate pre-mRNA to be processed into multiple mRNAs differing in their precise combination of exon sequences, resulting in the encoding of different protein isoforms. 2 Multiple modes of alternative splicing exist, such as alternative 5Ј or 3Јsplice-site usage, differential inclusion or skipping of particular exons, mutually exclusion of exons, and more. 3 Importantly, alternative splicing is often tightly regulated in a cell type-or developmental stage-specific mode. 3 The essential nature of this process is underscored by the fact that misregulation (missplicing events) is often related to human disease. 4 -6

Sox4-mediated caldesmon expression facilitates skeletal myoblast differentiation

“…Moreover, CaD expression induced by p53 suppresses Src-kinase-dependent podosome formation, which inhibits fibroblast and rat aortic SMC migration (Mukhopadhyay et al, 2009). By contrast, HeLa-l-CaD expression in endothelial cells and endothelial progenitor cells in the vasculature of various human tumors promotes cell migration for vasculogenesis and angiogenesis during tumor development (Zheng et al, 2004;Zheng et al, 2007). Of note, we observed that endogenous CaD in C2C12 myoblasts localized to the membrane ruffles, suggesting that CaD is involved in cell migration in C2C12 myoblasts.…”

Regulation of Microfilament Organization by Kaposi Sarcoma-associated Herpes Virus-cyclin·CDK6 Phosphorylation of Caldesmon

“…For instance, v-Src-transformed cells display a reduced expression of CALD1 (80,81), whereas v-ErbB2-transformed fibroblasts show enhanced tyrosine phosphorylation of CALD1 that correlates with stress fiber disassembly (82). Lastly, missplicing of the CALD1 gene has been observed in glioma microvasculature and is associated with tight junction breakdown between endothelial cells and vascular leakage (83). Thus, K-cyclin-induced phosphorylation of CALD1 could provide a cancer-promoting event, independent of, and in addition to, the impact of this cyclin on cell cycle progression and proliferation.…”

Histochemical Localization of Caldesmon in the CNS and Ganglia of the Mouse

“…This antibody was raised against a fragment of human l-caldesmon corresponding to amino acids (aa) 251-395, a sequence 93% identical between human and mouse (Basic Local Alignment Search Tool [BLAST] analysis; Altschul et al 1997) but both present in l-and h-caldesmon. This antibody was used by Zheng et al (2004) and Bogatcheva et al (2006). K. Agassandian and Cassell (2008) used a goat anti-caldesmon (N-19) antibody raised against a peptide mapping near the N-terminus of human caldesmon (sc-7575; Santa Cruz Biotechnology, Heidelberg, Germany).…”

“…Some studies show caldesmon only in blood vessels, others exclusively in neurons. In the normal human brain and in gliomas, caldesmon is expressed in endothelial cells, smcs, and pericytes of blood vessels; in the dura, it is expressed in fibroblasts (Zheng et al 2003;Zheng et al 2004). In the rat cortex and hippocampus, smcs of blood vessels and endothelial cells were reported to display caldesmon immunoreactivity (Kreipke et al 2006).…”

“…Cell culture experiments, however, demonstrated caldesmon in neurites and growth cones of cultured rat and chick neurons (Kira et al 1995;Alexanian et al 2001). Similarly, cultured astrocytes displayed caldesmon immunoreactivity (Abd-el-Basset et al 1991), but glial cells in tissue were not stained (Agassandian C et al 2000;Zheng et al 2004;Agassandian K and Cassell 2008).…”