Regulation of Transformed State by Calpastatin via PKCϵ in NIH3T3 Mouse Fibroblasts

doi:10.1006/bbrc.2001.6197

Biochemical and Biophysical Research Communications

Volume 290, Issue 1, 11 January 2002, Pages 510-517

https://doi.org/10.1006/bbrc.2001.6197 Get rights and content

Abstract

Ca²⁺-activated neutral protease calpain is ubiquitously expressed and may have pleiotropic biological functions. We have previously reported that repeated treatment of NIH3T3 mouse fibroblasts with the calpain inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN) resulted in the induction of transformed foci [T. Hiwasa, T. Sawada, and S. Sakiyama (1990) Carcinogenesis 11, 75–80]. To elucidate further the effects of calpain in malignant transformation of NIH3T3 cells, calpastatin, an endogenous specific inhibitor of calpain, was expressed in NIH3T3 cells by transfection with cDNA. G418-selected calpastatin-expressing clones showed a significant increase in the anchorage-independent growth ability. A similar increase in cloning efficiency in soft agar medium was also observed in calpain small-subunit-transfected clones. On the other hand, reduced expression of calpastatin achieved by transfection with calpastatin antisense cDNA in Ha-ras-transformed NIH3T3 (ras-NIH) cells caused morphological reversion as well as a decrease in anchorage-independent growth. When NIH3T3 cells were treated with ALLN for 3 days, cell growth was stimulated by approximately 10%. This growth stimulation by ALLN was not observed in ras-NIH cells, but recovered by expression of a dominant negative form of protein kinase C (PKC)ϵ but not by that of PKCα. Western blotting analysis showed that an increase in PKCϵ was much more prominent than that of PKCα in NIH3T3 cells after treatment with ALLN. These results are concordant with the notion that calpain suppresses malignant transformation by predominant degradation of PKCϵ.

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Citation Excerpt :
Thyrotropin-releasing hormone induces down-regulation of PKCε in pituitary cells, and the introduction of calpastatin into the cells inhibits this down-regulation, pointing to calpain being responsible for PKCε degradation (Eto et al., 1995). Calpain has been shown to suppress fibroblast malignant transformation by predominantly degrading PKCε (Hiwasa et al., 2002). Overall, the published and the present results indicate that in AD and in neuronal cells exposed to Aβ, calpain is responsible for PKCε degradation.
Amyloid β peptide (Aβ) plays a major role in the pathogenesis of Alzheimer's disease (AD). Aβ is toxic to neurons, possibly through causing initial synaptic dysfunction and neuronal membrane dystrophy, promoted by increased cellular Ca²⁺. Calpain (Ca²⁺-dependent protease) and caspase have been implicated in AD. Previously, we used calpain and caspase pharmacological inhibitors to study effects of Aβ25–35 (sAβ) on neuronal-like differentiated PC12 cells. We reported that sAβ-treated cells exhibited calpain activation and protein degradation (due to both calpain and caspase-8). We have now found that overexpression of the calpain specific inhibitor calpastatin in differentiated PC12 cells significantly inhibited the sAβ-induced calpain activation and decreased the protease activity. Calpastatin overexpression inhibited the sAβ-promoted degradation of fodrin, protein kinase Cε, β-catenin (membrane structural proteins and proteins involved in signal transduction pathways), and prevented the sAβ-induced alteration of neurite structure (manifested by varicosities). Overexpression of calpastatin also inhibited Ca²⁺-promoted calpain activation and protein degradation; this is consistent with the notion that the Aβ-induced increase in calpain activity results from a rise in cellular Ca²⁺, provided the calpastatin level is not so high as to strongly inhibit calpain. Carrying out transfection without selection allowed the comparison in the same culture of calpastatin-overexpressing with non-overexpressing cells. In cultures transfected with green fluorescent protein (GFP)–calpastatin plasmid, calpastatin overexpression (indicated by GFP-labeling) led to inhibition in sAβ-induced membrane propidium iodide (PI) permeability, whereas non-transfected, GFP-unlabeled cells exhibited PI permeability. Overall, the results demonstrate that the effects of Aβ-toxicity studied here were attenuated to a large extent by calpastatin overexpression, indicating that the protease calpain is involved in Aβ-toxicity (obviating a primary, direct role for caspases). Increased expression of calpastatin and/or decrease in calpain may serve as one of the means for ameliorating some of the early symptoms of AD.
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^☆: Abbreviations used: ALLN/calpain inhibitor I, N-acetyl-Leu-Leu-norleucinal; Dex, dexamethasone; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; MTT, 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide; PI3K, phosphatidylinositol 3-kinase; PIP₂, phosphatidylinositol-3,4-bisphosphate; PIP₃, phosphatidylinositol-3,4,5-trisphosphate; PKC, protein kinase C.

¹: To whom correspondence and reprint requests should be addressed. Fax: 81 (43) 226-2037. E-mail: [email protected].

²: Present address: Department of Pediatric Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan.

³: Present address: Department of Neurology, Graduate School of Medicine, Chiba University, Chiba, Japan.

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Regular ArticleRegulation of Transformed State by Calpastatin via PKCϵ in NIH3T3 Mouse Fibroblasts☆

Abstract

Biochem. Biophys. Res. Commun.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Immunol. Method

FEBS Lett.

FEBS Lett.

Biochem. Biophys. Res. Commun.

FEBS Lett.

J. Biol. Chem.

Cell

Calpain: Novel family members, activation, and physiologic function

Biol. Chem. Hoppe–Seyler

Calpain superfamily

Biochem. J.

Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus

Nat. Genet.

Regular Article
Regulation of Transformed State by Calpastatin via PKCϵ in NIH3T3 Mouse Fibroblasts☆