Overexpression of Calreticulin Modulates Protein Kinase B/Akt Signaling to Promote Apoptosis during Cardiac Differentiation of Cardiomyoblast H9c2 Cells

Abstract

Calreticulin is a Ca2؉ -binding molecular chaperone of the lumen of the endoplasmic reticulum. Calreticulin has been shown to be essential for cardiac and neural development in mice, but the mechanism by which it functions in cell differentiation is not fully understood. To examine the role of calreticulin in cardiac differentiation, the calreticulin gene was introduced into rat cardiomyoblast H9c2 cells, and the effect of calreticulin overexpression on cardiac differentiation was examined. Upon culture in a differentiation medium containing fetal calf serum (1%) and retinoic acid (10 nM), cells transfected with the calreticulin gene were highly susceptible to apoptosis compared with controls. In the gene-transfected cells, protein kinase B/Akt signaling was significantly suppressed during differentiation. Furthermore, protein phosphatase 2A, a Ser/Thr protein phosphatase, was significantly up-regulated, implying suppression of Akt signaling due to dephosphorylation of Akt by the up-regulated protein phosphatase 2A via regulation of Ca 2؉ homeostasis. Thus, overexpression of calreticulin promotes differentiation-dependent apoptosis in H9c2 cells by suppressing the Akt signaling pathway. These findings indicate a novel mechanism by which cytoplasmic Akt signaling is modulated to cause apoptosis by a resident protein of the endoplasmic reticulum, calreticulin. Calreticulin (CRT)1 is a Ca 2ϩ -binding molecular chaperone in the endoplasmic reticulum (ER) (1). It is a highly conserved protein with Ͼ90% amino acid identity in mammals, including human, rabbit, rat, and mouse (2). The gene has also been found in insects, nematodes, protozoa, and plants, but not in yeast or prokaryotes (1, 3), suggesting a general function in living cells. CRT is involved in many biological process, including regulation of Ca 2ϩ homeostasis and intracellular signaling, cell adhesion, gene expression, and glycoprotein folding (3, 4) and nuclear transport (5).CRT is well expressed in embryonic rat heart, but its expression is significantly suppressed after birth (6). Cardiac development is believed to be regulated cooperatively by a variety of proteins, including signaling molecules (e.g. fibroblast growth factor, transforming growth factor-␤, ErbB2/B4, neuregulin, etc.), cell adhesion molecules (e.g. vascular cell adhesion molecule, ␣ 4 integrin, and versican), ion channels, and transcription factors (e.g. GATA, myocyte enhancer factor-2, HAND, chicken ovalbumin upstream promoter transcription factor II, Nkx2.5, TBX5, NF-ATc, Smad6, Pax3, retinoid X receptor/retinoic acid receptor, TEF-1, WT-1, etc.) (7). Interestingly, CRT gene expression is known to be regulated by a transcription factor (Nkx2.5) that is involved in the regulation of gene expression for cardiac development (8). Recently, it has been shown that CRT is essential for cardiac and neural development in mice (9, 10). CRT-deficient embryonic cells show impaired nuclear import of the transcription factor NF-AT3 (nuclear factor of activated T cells), indicating that CRT function...

Calreticulin promotes cell motility and enhances resistance to anoikis through STAT3–CTTN–Akt pathway in esophageal squamous cell carcinoma

“…However, in human glioblastoma U251MG cells, overexpression of CRT-enhanced radiation-induced apoptosis by suppressing Akt signaling (Okunaga et al, 2006). Elevation of CRT promoted apoptosis during cardiac differentiation by negatively regulating Akt activation by upregulating the serine/threonine phosphatase PP2A (Kageyama et al, 2002). In mature cardiomyocytes, enhanced CRT expression disrupted intracellular calcium regulation, leading to calcium-dependent apoptosis (Lim et al, 2008).…”

“…Recent evidence has shown that CRT protects fibroblasts from anoikis through binding to LRP1 and TSP-1 (Pallero et al, 2008). Akt activation or inactivation has been reported to play an important role in CRT-associated apoptosis (Kageyama et al, 2002;Okunaga et al, 2006;Lim et al, 2008). CRT-deficient mice show embryonic lethal characteristics due to changes in cell adhesiveness and impairment of cardiac development and function (Coppolino et al, 1997;Rauch et al, 2000;Mesaeli and Phillipson, 2004).…”

Functional Linkage between the Endoplasmic Reticulum Protein Hsp47 and Procollagen Expression in Human Vascular Smooth Muscle Cells

“…This is followed by receptor clustering and autophosphorylation and activation of JNKs (50). Recently, overexpression of calreticulin in myoblasts was associated with up-regulation of protein phosphatase 2A, further highlighting the potential for ER events to be transduced outside that compartment (51).…”

Molecular basis of angiogenesis and cancer

“…1995;Kageyama et al, 2002). It has been shown to be essential for cardiac and neural development in mice, but the mechanism by which it functions in cell differentiation is not fully understood.…”