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Biomedical subjects

Marek Michalak

Publications and source records attributed to Marek Michalak.

At least 19 recordsLinked to original sources
PubMed2006

Ultrastructural analysis of development of myocardium in calreticulin-deficient mice.

BACKGROUND: Calreticulin is a Ca2+ binding chaperone of the endoplasmic reticulum which influences gene expression and cell adhesion. The levels of both vinculin and N-cadherin are induced by calreticulin expression, which play important roles in cell adhesiveness. Cardiac development is strictly dependent upon the ability of cells to adhere to their substratum and to communicate with their neighbours. RESULTS: We show here that the levels of N-cadherin are downregulated in calreticulin-deficient mouse embryonic hearts, which may lead to the disarray and wavy appearance of myofibrils in these mice, which we detected at all investigated stages of cardiac development. Calreticulin wild type mice exhibited straight, thick and abundant myofibrils, which were in stark contrast to the thin, less numerous, disorganized myofibrils of the calreticulin-deficient hearts. Interestingly, these major differences were only detected in the developing ventricles while the atria of both calreticulin phenotypes were similar in appearance at all developmental stages. Glycogen also accumulated in the ventricles of calreticulin-deficient mice, indicating an abnormality in cardiomyocyte metabolism. CONCLUSION: Calreticulin is temporarily expressed during heart development where it is required for proper myofibrillogenesis. We postulate that calreticulin be considered as a novel cardiac fetal gene.

Animals↗
PubMed2006

Caspase 12 in calnexin-deficient cells.

We investigated a role for calnexin, caspase 12, and Bap31 in endoplasmic reticulum stress-induced apoptosis in calnexin-deficient mouse embryonic fibroblasts and a calnexin-deficient human T cell line (NKR). We showed that calnexin-deficient mouse embryonic fibroblasts are relatively resistant to endoplasmic reticulum stress-induced apoptosis. Western blot analysis demonstrated that both wild-type and calnexin-deficient cells contained a caspase 12 protein. Caspase 12 expression was slightly inhibited in calnexin-deficient cells, and the protein carried out specific cleavage in the presence of thapsigargin. Immunoprecipitation experiments revealed that in the endoplasmic reticulum, caspase 12 forms complexes with Bap31 and calnexin. Treatment of wild-type cells with thapsigargin induced apoptosis and cleavage of Bap31. However, in the absence of calnexin, there was no significant cleavage of Bap31. There was also a negligible processing of caspase 8 in these cells. This work indicates that calnexin may play a role in modulating the sensitivity of a cell to apoptosis induced by endoplasmic reticulum stress, in conjunction with caspase 12 and Bap31.

Animals↗
PubMed2006

Regulation of peripheral T cell activation by calreticulin.

Regulated expression of positive and negative regulatory factors controls the extent and duration of T cell adaptive immune response preserving the organism's integrity. Calreticulin (CRT) is a major Ca2+ buffering chaperone in the lumen of the endoplasmic reticulum. Here we investigated the impact of CRT deficiency on T cell function in immunodeficient mice reconstituted with fetal liver crt-/- hemopoietic progenitors. These chimeric mice displayed severe immunopathological traits, which correlated with a lower threshold of T cell receptor (TCR) activation and exaggerated peripheral T cell response to antigen with enhanced secretion of inflammatory cytokines. In crt-/- T cells TCR stimulation induced pulsatile cytosolic elevations of Ca2+ concentration and protracted accumulation of nuclear factor of activated T cells in the nucleus as well as sustained activation of the mitogen-activated protein kinase pathways. These observations support the hypothesis that CRT-dependent shaping of Ca2+ signaling critically contributes to the modulation of the T cell adaptive immune response.

Animals↗
PubMed2006

Assembling pieces of the cardiac puzzle; calreticulin and calcium-dependent pathways in cardiac development, health, and disease.

Calreticulin is a Ca(2+)-binding chaperone of the sarcoplasmic/endoplasmic reticulum. It is an important Ca(2+) buffer, a regulator of Ca(2+) homeostasis, and a component of protein quality control processes in the secretory pathway. Calreticulin is essential for cardiac development; its gene is tightly regulated during cardiogenesis, and in the absence of calreticulin, cardiac development is impaired. The protein is highly expressed in the developing heart and down-regulated after birth in the healthy mature heart. Overexpression of calreticulin in postnatal heart leads to bradyarrhythima and complete heart block, followed by sudden death. The calreticulin gene is a target of transcription factors involved in fetal cardiac program (Nkx2.5, myocardin, myocyte enhancer factor 2C, and GATA6). Calreticulin works upstream of calcineurin and myocyte enhancer factor 2C in a Ca(2+)-dependent signal transduction cascade linking the endoplasmic reticulum and the nucleus during cardiac development.

Animals↗
PubMed2006

Overview of the role for calreticulin in the enhancement of wound healing through multiple biological effects.

Calreticulin (CRT), an intracellular chaperone protein crucial for the proper folding and transport of proteins through the endoplasmic reticulum, has more recent acclaim as a critical regulator of extracellular functions, particularly in mediating cellular migration and as a requirement for phagocytosis of apoptotic cells. Consistent with these functions, we show that the topical application of CRT has profound effects on the process of wound healing by causing a dose-dependent increase in epithelial migration and granulation tissue formation in both murine and porcine normal and impaired animal models of skin injury. These effects of CRTare substantiated, in vitro, as we show that CRT strongly induces cell migration/wound closure of human keratinocytes and fibroblasts, using a wound/scratch plate assay, and stimulates cellular proliferation of human keratinocytes, fibroblasts, and vascular endothelial cells, providing mechanistic insight into how CRT functions in repair. Similarly, in both animal models, the histology of the wounds show marked proliferation of basal keratinocytes and dermal fibroblasts, dense cellularity of the dermis with notably increased numbers of macrophages and well-organized collagen fibril deposition. Thus, CRT profoundly affects the wound healing process by recruiting cells essential for repair into the wound, stimulating cell growth, and increasing extracellular matrix production.

Animals↗
PubMed2005

Identification by mutational analysis of amino acid residues essential in the chaperone function of calreticulin.

Calreticulin is a Ca2+ -binding chaperone that resides in the lumen of the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and in the folding of newly synthesized glycoproteins. In this study, we have used site-specific mutagenesis to map amino acid residues that are critical in calreticulin function. We have focused on two cysteine residues (Cys(88) and Cys(120)), which form a disulfide bridge in the N-terminal domain of calreticulin, on a tryptophan residue located in the carbohydrate binding site (Trp(302)), and on certain residues located at the tip of the "hairpin-like" P-domain of the protein (Glu(238), Glu(239), Asp(241), Glu(243), and Trp(244)). Calreticulin mutants were expressed in crt(-/-) fibroblasts, and bradykinin-dependent Ca2+ release was measured as a marker of calreticulin function. Bradykinin-dependent Ca2+ release from the endoplasmic reticulum was rescued by wild-type calreticulin and by the Glu(238), Glu(239), Asp(241), and Glu(243) mutants. The Cys(88) and Cys(120) mutants rescued the calreticulin-deficient phenotype only partially ( approximately 40%), and the Trp(244) and Trp(302) mutants did not rescue it at all. We identified four amino acid residues (Glu(239), Asp(241), Glu(243), and Trp(244)) at the hairpin tip of the P-domain that are critical in the formation of a complex between ERp57 and calreticulin. Although the Glu(239), Asp(241), and Glu(243) mutants did not bind ERp57 efficiently, they fully restored bradykinin-dependent Ca2+ release in crt(-/-) cells. This indicates that binding of ERp57 to calreticulin may not be critical for the chaperone function of calreticulin with respect to the bradykinin receptor.

Animals↗
PubMed2005

Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte.

Apoptotic-cell removal is critical for development, tissue homeostasis, and resolution of inflammation. Although many candidate systems exist, only phosphatidylserine has been identified as a general recognition ligand on apoptotic cells. We demonstrate here that calreticulin acts as a second general recognition ligand by binding and activating LDL-receptor-related protein (LRP) on the engulfing cell. Since surface calreticulin is also found on viable cells, a mechanism preventing inadvertent uptake was sought. Disruption of interactions between CD47 (integrin-associated protein) on the target cell and SIRPalpha (SHPS-1), a heavily glycosylated transmembrane protein on the engulfing cell, permitted uptake of viable cells in a calreticulin/LRP-dependent manner. On apoptotic cells, CD47 was altered and/or lost and no longer activated SIRPalpha. These changes on the apoptotic cell create an environment where "don't eat me" signals are rendered inactive and "eat me" signals, including calreticulin and phosphatidylserine, congregate together and signal for removal.

Animals↗
PubMed2005

Calreticulin signals upstream of calcineurin and MEF2C in a critical Ca(2+)-dependent signaling cascade.

We uncovered a new pathway of interplay between calreticulin and myocyte-enhancer factor (MEF) 2C, a cardiac-specific transcription factor. We establish that calreticulin works upstream of calcineurin and MEF2C in a Ca(2+)-dependent signal transduction cascade that links the endoplasmic reticulum and the nucleus during cardiac development. In the absence of calreticulin, translocation of MEF2C to the nucleus is compromised. This defect is reversed by calreticulin itself or by a constitutively active form of calcineurin. Furthermore, we show that expression of the calreticulin gene itself is regulated by MEF2C in vitro and in vivo and that, in turn, increased expression of calreticulin affects MEF2C transcriptional activity. The present findings provide a clear molecular explanation for the embryonic lethality observed in calreticulin-deficient mice and emphasize the importance of calreticulin in the early stages of cardiac development. Our study illustrates the existence of a positive feedback mechanism that ensures an adequate supply of releasable Ca(2+) is maintained within the cell for activation of calcineurin and, subsequently, for proper functioning of MEF2C.

Animals↗
PubMed2005

Endoplasmic reticulum quality control and apoptosis.

The ER is one of the most important folding compartments within the cell, as well as an intracellular Ca(2+) storage organelle and it contains a number of Ca(2+) regulated molecular chaperones responsible for the proper folding of glycosylated as well as non-glycosylated proteins. The luminal environment of the ER contains Ca(2+) which is involved in regulating chaperones such as calnexin and calreticulin, as well as apoptotic proteins caspase-12 and Bap31, which may play an important role in determining cellular sensitivity to ER stress and apoptosis. The ER quality control system consists of several molecular chaperones, including calnexin, that assist in properly folding proteins and transporting them through the ER as well as sensing misfolded proteins, attempting to refold them and if this is not possible, targeting them for degradation. Accumulation of misfolded protein in the ER leads to activation of genes responsible for the expression of ER chaperones. The UPR mechanism involves transcriptional activation of chaperones by the membrane-localized transcription factor ATF6, in conjunction with the ER membrane kinase IRE1, as well as translational repression of protein synthesis by another ER membrane kinase PERK. When accumulation of misfolded protein becomes toxic, apoptosis is triggered, potentially with IRE1 involved in signaling via caspase-12. Both the extrinsic and intrinsic apoptotic pathways appear to culminate in the activation of caspases and this results in the recruitment of mitochondria in an essential amplifying manner. Bap31 may direct pro-apoptotic crosstalk between the ER and the mitochondria via Ca(2+) in conjunction with caspase-12 and calnexin. Accordingly, ER stress and the resultant Ca(2+) release must be very carefully regulated because of their effects in virtually all areas of cell function.

Animals↗
PubMed2005

Impaired cytolytic activity in calreticulin-deficient CTLs.

Calreticulin is an endoplasmic reticulum-resident chaperone that is stored in the cytotoxic granules of CTLs and NK cells and is released with granzymes and perforin upon recognition of target cells. To investigate the role of calreticulin in CTL-mediated killing, we generated CTL lines from crt(+/+) and crt(-/-) mice expressing a constitutively active form of calcineurin in the heart. Crt(-/-) CTLs showed reduced cytotoxic activity toward allogeneic target cells despite normal production, intracellular localization, and activity of granzymes and despite perforin overexpression. Comparable or higher amounts of granzymes were degranulated by crt(-/-) cells in response to immobilized anti-CD3 Abs, indicating that calreticulin is dispensable for the signal transduction that leads to granule exocytosis. The ability to form conjugates with target cells was affected in the crt(-/-) CTLs, explaining the observed reduction in cytotoxicity. Conjugate formation and cytotoxicity were completely restored by treatments that facilitate recognition and contact with target cells, a prerequisite for degranulation and killing. Therefore, we conclude that calreticulin is dispensable for the cytolytic activity of granzymes and perforin, but it is required for efficient CTL-target cell interaction and for the formation of the death synapse.

Animals↗
PubMed2005

Initiation of embryonic cardiac pacemaker activity by inositol 1,4,5-trisphosphate-dependent calcium signaling.

In the adult, the heart rate is driven by spontaneous and repetitive depolarizations of pacemaker cells to generate a firing of action potentials propagating along the conduction system and spreading into the ventricles. In the early embryo before E9.5, the pacemaker ionic channel responsible for the spontaneous depolarization of cells is not yet functional. Thus the mechanisms that initiate early heart rhythm during cardiogenesis are puzzling. In the absence of a functional pacemaker ionic channel, the oscillatory nature of inositol 1,4,5-trisphosphate (InsP3)-induced intracellular Ca2+ signaling could provide an alternative pacemaking mechanism. To test this hypothesis, we have engineered pacemaker cells from embryonic stem (ES) cells, a model that faithfully recapitulates early stages of heart development. We show that InsP3-dependent shuttle of free Ca2+ in and out of the endoplasmic reticulum is essential for a proper generation of pacemaker activity during early cardiogenesis and fetal life.

Animals↗
PubMed2005

Calreticulin, a Ca2+-binding chaperone of the endoplasmic reticulum.

Calreticulin is a 46-kDa Ca2+-binding chaperone found across a diverse range of species. The protein is involved in the regulation of intracellular Ca2+ homeostasis and endoplasmic reticulum (ER) Ca2+ storage capacity. Calreticulin is also an important molecular chaperone involved in "quality control" within secretory pathways. The protein contains structurally and functionally unique domains with specialized functions. Studies on calreticulin knockout mice indicate that the protein is essential in early cardiac development. The protein also plays an important role in autoimmunity and cancer.

Animals↗
PubMed2005

Cellular functions of endoplasmic reticulum chaperones calreticulin, calnexin, and ERp57.

Glycosylated proteins destined for the cell surface or to be secreted from the cell are trafficked through the endoplasmic reticulum during synthesis and folding. Correct folding is determined in large part by the sequence of the protein, but it is also assisted by interaction with enzymes and chaperones of the endoplasmic reticulum. Calreticulin, calnexin, and ERp57 are among the endoplasmic chaperones that interact with partially folded glycoproteins and determine if the proteins are to be released from the endoplasmic reticulum to be expressed, or alternatively, if they are to be sent to the proteosome for degradation. Studies on the effect of alterations in the expression and function of these proteins are providing information about the importance of this quality control system, as well as uncovering other important functions these proteins play outside of the endoplasmic reticulum.

Animals↗
PubMed2004

Calreticulin, Ca2+, and calcineurin - signaling from the endoplasmic reticulum.

Calcium (Ca2+) is a universal signalling molecule involved in many aspects of cellular function. The majority of intracellular Ca2+ is stored in the endoplasmic reticulum and once Ca2+ is released from the endoplasmic reticulum, specific plasma membrane Ca2+ channels are activated, resulting in increased intracellular Ca2+. In the lumen of the endoplasmic reticulum, Ca2+ is buffered by Ca2+ binding chaperones such as calreticulin. Calreticulin-deficiency is lethal in utero due to impaired cardiac development and in the absence of calreticulin, Ca2+ storage capacity within the endoplasmic reticulum and inositol 1,4,5-trisphosphate (InsP3) receptor mediated Ca2+ release from the endoplasmic reticulum are compromised. Over-expression of constitutively active calcineurin in the heart rescues calreticulin-deficient mice from embryonic lethality. This observation indicates that calreticulin is a key upstream regulator of calcineurin in Ca2+-signalling pathways and highlights the importance of the endoplasmic reticulum and endoplasmic reticulum-dependent Ca2+ homeostasis for cellular commitment and tissue development during organogenesis. Furthermore, Ca2+ handling by the endoplasmic reticulum has profound effects on cell sensitivity to apoptosis. Signalling between calreticulin in the lumen of the endoplasmic reticulum and calcineurin in the cytoplasm may play a role in the modulation of cell sensitivity to apoptosis and the regulation of Ca2+-dependent apoptotic pathways.

Animals↗
PubMed2004

Contrasting functions of calreticulin and calnexin in glycoprotein folding and ER quality control.

Calreticulin and calnexin are homologous lectins that serve as molecular chaperones for glycoproteins in the endoplasmic reticulum of eukaryotic cells. Here we show that calreticulin depletion specifically accelerates the maturation of cellular and viral glycoproteins with a modest decrease in folding efficiency. Calnexin depletion prevents proper maturation of some proteins such as influenza hemagglutinin but does not interfere appreciably with the maturation of several others. A dramatic loss of stringency in the ER quality control with transport at the cell surface of misfolded glycoprotein conformers is only observed when substrate access to both calreticulin and calnexin is prevented. Although not fully interchangeable during assistance of glycoprotein folding, calreticulin and calnexin may work, independently, as efficient and crucial factors for retention in the ER of nonnative polypeptides.

Animals↗
PubMed2004

Competition of CUGBP1 and calreticulin for the regulation of p21 translation determines cell fate.

Induction of p21 in senescent human fibroblasts plays a key role in the inactivation of cyclin-dependent kinases and the resulting irreversible growth arrest in the early stages of cell senescence. We found that RNA-binding proteins are critical regulators of p21 during senescence. Two RNA-binding proteins, CUGBP1 and calreticulin (CRT), interact with the same nucleotide sequences within the 5' region of p21 mRNA, but have opposite effects on the translation of p21 mRNA. CUGBP1 increases translation of p21 mRNA, whereas CRT blocks translation of p21 via stabilization of a stem-loop structure within the 5' region of the p21 mRNA. CUGBP1 and CRT compete for binding to p21 mRNA and thereby the regulation of p21 translation. In senescent fibroblasts, CUGBP1 displaces CRT from the p21 mRNA and releases CRT-dependent repression of p21 translation leading to growth arrest and development of a senescent phenotype. These data present evidence that competition between RNA-binding proteins for the regulation of p21 translation determines cell fate.

5' Untranslated Regions↗
PubMed2004

Calreticulin in the heart.

Calreticulin is a Ca2+ binding/storage chaperone resident protein of the endoplasmic reticulum. This protein plays a key role in the calreticulin/calnexin cycle and the quality control pathways in the endoplasmic reticulum. Calreticulin deficiency is lethal due to impaired cardiac development. However, over-expression of the protein in developing and postnatal heart leads to bradycardia, complete heart block and sudden death. Ultrastructural evidence indicates that the deficiency associated with the absence of calreticulin in the heart may be due to a defect in the development of the contractile apparatus and/or a defect in development of the conductive system as well as a metabolic abnormality. Collectively, we postulate that calreticulin and endoplasmic reticulum plays an important role in cardiac development and postnatal pathologies.

Animals↗