Chronophin regulates active vitamin B6 levels and transcriptomic features of glioblastoma cell lines cultured under non-adherent, serum-free conditions

Abstract

BackgroundThe phosphatase chronophin (CIN/PDXP) has been shown to be an important regulator of glioma cell migration and invasion. It has two known substrates: p-Ser3-cofilin, the phosphorylated form of the actin binding protein cofilin, and pyridoxal 5′-phosphate, the active form of vitamin B6. Phosphoregulation of cofilin, among other functions, plays an important role in cell migration, whereas active vitamin B6 is a cofactor for more than one hundred enzymatic reactions. The role of CIN has yet only been examined in glioblastoma cell line models derived under serum culture conditions.ResultsWe found that CIN is highly expressed in cells cultured under non-adherent, serum-free conditions that are thought to better mimic the in vivo situation. Furthermore, the substrates of CIN, p-Ser3-cofilin and active vitamin B6, were significantly reduced as compared to cell lines cultured in serum-containing medium. To further examine its molecular role we stably knocked down the CIN protein with two different shRNA hairpins in the glioblastoma cell lines NCH421k and NCH644. Both cell lines did not show any significant alterations in proliferation but expression of differentiation markers (such as GFAP or TUBB3) was increased in the knockdown cell lines. In addition, colony formation was significantly impaired in NCH644. Of note, in both cell lines CIN knockdown increased active vitamin B6 levels with vitamin B6 being known to be important for S-adenosylmethionine biosynthesis. Nevertheless, global histone and DNA methylation remained unaltered as was chemoresistance towards temozolomide. To further elucidate the role of phosphocofilin in glioblastoma cells we applied inhibitors for ROCK1/2 and LIMK1/2 to our model. LIMK- and ROCK-inhibitor treatment alone was not toxic for glioblastoma cells. However, it had profound, but antagonistic effects in NCH421k and NCH644 under chemotherapy.ConclusionIn non-adherent glioblastoma cell lines cultured in serum-free medium, chronophin knockdown induces phenotypic changes, e.g. in colony formation and transcription, but these are highly dependent on the cellular background. The same is true for phenotypes observed after treatment with inhibitors for kinases regulating cofilin phosphorylation (ROCKs and LIMKs). Targeting the cofilin phosphorylation pathway might therefore not be a straightforward therapeutic option in glioblastoma.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4440-4) contains supplementary material, which is available to authorized users.

Potential Antimetastatic Effect of Timosaponin AIII against Human Osteosarcoma Cells through Regulating the Integrin/FAK/Cofilin Axis

“…The activation of cofilin is regulated through phosphorylation by cofilin-binding molecules. Phosphorylation at Ser-3 by LIMKs and TESKs leads to the deactivation of cofilin; by contrast, dephosphorylation by SSHs and PDXP leads to the reactivation of cofilin [ 16 , 29 ]. TESKs are related to LIMKs and contain the highly homologues kinase domain of LIMKs at the N-terminus and a unique C-terminal proline-rich domain.…”

“…The a of cofilin is regulated through phosphorylation by cofilin-binding molecules. Ph lation at Ser-3 by LIMKs and TESKs leads to the deactivation of cofilin; by dephosphorylation by SSHs and PDXP leads to the reactivation of cofilin [16,29 are related to LIMKs and contain the highly homologues kinase domain of LIM N-terminus and a unique C-terminal proline-rich domain. TESK1 acts downstre tegrins and plays a critical role in integrin-mediated actin reorganization by ph lating and deactivating cofilin [30].…”

“…When phosphorylated at serine 3, cofilin is deactivated and cannot bind to actin molecules, thus impairing cytoskeleton remodelling. Cofilin Ser3 is activated through dephosphorylation by slingshot protein phosphatase (SSH) and pyridoxal phosphatase (PDXP) [ 14 , 15 , 16 ].…”

MicroRNA‑181 inhibits glioblastoma cell growth by directly targeting CCL8

“…In recent years, with the development of molecular biology techniques, tumor-related genetic research has increased (8). At present, gene therapies and molecular-targeted drugs are a novel area of study (9,10). The most important aspect of these treatments is identifying a specific molecular therapy target for different tumors.…”

CircularLRRC7 is a Potential Tumor Suppressor Associated With miR-1281 and PDXP Expression in Glioblastoma

“…GBM patients with lower median PDXP expression levels had significantly shorter mean survival periods ( Schulze et al, 2016 ). In addition, PDXP regulated active vitamin B6 levels and transcriptomic features of GBM cell lines cultured under non-adherent, serum-free conditions ( Schulze et al, 2018 ), indicating a potential role of PDXP in tumor metabolism and cancer stem cells. Future studies will further unveil the roles of circLRRC7, miR-1281, and PDXP in the development of GBM and verify the prognostic values of these genes in GBM patients.…”