Apoptosis (2019) 24:245–255 https://doi.org/10.1007/s10495-019-01532-0 REVIEW Calreticulin in phagocytosis and cancer: opposite roles in immune response outcomes Alejandro Schcolnik‑Cabrera1 · Bernardo Oldak3 · Mandy Juárez1 · Mayra Cruz‑Rivera2 · Ana Flisser2 · Fela Mendlovic2,3 Published online: 30 March 2019 © Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract Calreticulin (CRT) is a pleiotropic and highly conserved molecule that is mainly localized in the endoplasmic reticulum. Recently, CRT has gained special interest for its functions outside the endoplasmic reticulum where it has immunomodula- tory properties. CRT translocation to the cell membrane serves as an “eat me” signal and promotes efferocytosis of apoptotic cells and cancer cell removal with completely opposite outcomes. Efferocytosis results in a silenced immune response and homeostasis, while removal of dying cancer cells brought about by anthracycline treatment, ionizing-irradiation or photo- dynamic therapy results in immunogenic cell death with activation of the innate and adaptive immune responses. In addi- tion, CRT impacts phagocyte activation and cytokine production. The effects of CRT on cytokine production depend on its conformation, species specificity, degree of oligomerization and/or glycosylation, as well as its cellular localization and the molecular partners involved. The controversial roles of CRT in cancer progression and the possible role of the CALR gene mutations in myeloproliferative neoplasms are also addressed. The release of CRT and its influence on the different cells involved during efferocytosis and immunogenic cell death points to additional roles of CRT besides merely acting as an “eat me” signal during apoptosis. Understanding the contribution of CRT in physiological and pathological processes could give us some insight into the potential of CRT as a therapeutic target. Keywords Calreticulin · Apoptosis · Phagocytosis · Cancer · Immunogenic cell death · Immune response Introduction involved in Ca2+ homeostasis; it regulates diverse cellular functions and acts as a lectin-like chaperone with specificity Calreticulin (CRT) is a 46 kDa soluble protein that local- for monoglucosylated oligosaccharide in asparagine-linked izes primarily to the endoplasmic reticulum (ER) and is glycoproteins that contributes to their correct folding in the highly conserved in all species. Two of the major functions ER [1–3]. The structure of CRT comprises three domains: of the ER are Ca 2+ sequestration and glycoprotein assem- The N-terminal globular domain, a flexible proline-rich P bly and secretion; CRT has a crucial role in both. CRT is intermediate arm-like domain and a C domain at the car- boxyl terminal. The N terminal of the molecule is predicted to be a highly folded globular structure, with eight anti-par- Schcolnik-Cabrera Alejandro and Oldak Bernardo contributed allel β sheets connected by loops, its amino acid sequence equally to the present work. is extremely conserved and binds Zn2+ [4]. It contains three cysteine residues conserved from higher plants to humans * Fela Mendlovic [email protected] that are responsible for the correct folding of the protein. This domain can interact with α integrins and with the DNA 1 División de Investigación Básica, Instituto Nacional de binding site of steroid receptors [5, 6]. The P domain con- Cancerología, Ciudad de México, Mexico sists of an extended region stabilized by three anti-parallel 2 Departamento de Microbiología y Parasitología, Facultad β strands [7] and includes a proline-rich sequence with two de Medicina, Universidad Nacional Autónoma de México, sets of three highly conserved repeats. This region binds Ciudad de México, Mexico 2+ Ca with high affinity (K = 1 μM), and together with the 3 d Facultad de Ciencias de la Salud, Universidad Anáhuac N domain, is essential for the lectin-like chaperone function México Norte, Huixquilucan, Estado De México, Mexico Vol.:(0123456789)1 3 246 Apoptosis (2019) 24:245–255 [3]. The C domain binds Ca 2+ with high capacity due to condensation and biochemical alterations such as activation its acidic nature, with more than 35 aspartic and glutamic of proteases and fragmentation of DNA [11]. The uptake acid residues, which function as multiple low-affinityCa 2+ and removal of apoptotic cells by phagocytes, or efferocytic binding sites and terminates with the ER retrieval signal [8, cells, is known as “efferocytosis”, and is driven through 9] (Fig. 1). several signals that flag the dying cell to be recognized With the discovery of CRT in compartments beyond the and engulfed [12]. In fact, early in the apoptotic program, ER, immunogenic roles and additional functions have been cells display molecular “eat me” signals on their surface to revealed. Among the functions that have gained particular alert phagocytes that they must be removed [11]. Nowa- interest in the last years are its role in promoting efferocyto- days, CRT is recognized as one of such signals. The first sis, in the clearance of dying cells by macrophages, and in report about CRT involvement in apoptosis was published the immunogenic cell death (ICD) of cancer cells induced by Ogden et al., who described that CRT has an important under specific treatments. In the present paper, the recent role in apoptotic Jurkat T cell clearance, driven by its ability findings on the role of CRT during these crucial functions of to bind to the innate immune protein CD91 (also known as myeloid-derived monocytic cells will be addressed. the low-density protein receptor-related protein, LRP-1), as well as to C1q and the mannose-binding lectin (MBL) [13]. Although C1q and MBL are involved in complement acti- Calreticulin and efferocytosis vation, they also attach to apoptotic cells by their globular domains to facilitate the ingestion by macrophages. After Phagocytosis is an essential process for a wide variety of C1q or MBL attachment, CRT binds to the collagenous entities, from unicellular organisms that use it as a means tails of both proteins, and to CD91 on the macrophage cell for nutrition, to multicellular organisms that depend on surface to promote the engagement of this receptor. These this complex process for correct function of the immune series of events initiate macropinocytosis that results in the response through the activation of phagocytes. Moreover, engulfment of the apoptotic cell [13]. CRT functions at both phagocytosis is important for resolution of inflammation via sides of the phagocytic or efferocytic synapse, at the surface the uptake of apoptotic cells, as well as for the maintenance of the apoptotic cell ready to be engulfed, as well as on of tissue homeostasis and protection against neoplastic cells. the phagocyte membrane and in the milieu surrounding the Failure in the clearance of apoptotic cells has been associ- phagocytic synapse. ated with autoimmunity, as it leads to pathologic inflamma- tory responses and induction of proinflammatory cytokine CRT on the apoptotic cell surface release [10]. Apoptosis, or programed cell death, is an ATP-depend- Phosphatidylserine (PS) is one of the best characterized mol- ent cellular process characterized by certain morpho- ecules that mediate phagocytosis, located in the inner leaflet logical changes, including membrane blebbing, nuclear of the cell membrane of living cells. Shortly upon induction Fig. 1 CRT structure and domain interactions. CRT has three structural domains, each one interacts with different molecular partners and has diverse functions 1 3 Apoptosis (2019) 24:245–255 247 of apoptosis, PS translocates to the outer leaflet of the cell with a decrease in CD47 [26], a molecule whose interac- membrane and serves as an “eat me signal”. CRT, normally tion with signal regulatory protein (SIRP)-α (also known localized in the ER lumen, also translocates to the outer cell as CD172a) in macrophages prevents phagocytosis of via- membrane where, along with PS, promotes phagocytosis of ble cells, through the activation of the immunoreceptor apoptotic cells but not its adhesion to phagocytes. Recently, tyrosine-based inhibition motifs (ITIMs) [27]. In addition, CRT on apoptotic cells was shown to bind to PS through its engulfment of viable cells, in particular neutrophils, by a C-terminal acidic domain in a calcium-dependent manner, mechanism termed programmed cell phagocytosis (PrCP) and serves as a PS-anchor factor that cooperates with other has been reported. The authors showed that macrophages PS-binding molecules, such as C1q [14, 15]. Induction of are the source of CRT on the surface of the living neutro- apoptosis by UVB irradiation results in the mobilization phils to be engulfed, and that CRT binds to asialoglycans of CRT to the outer cell membrane even before PS trans- exposed on the neutrophil surface [25, 28]. These findings location [16]. CRT also interacts with C1q for the uptake suggest that PrCP could be a more general phenomenon of apoptotic cells [17–19]. The globular domain of CRT involving other cell types involved in tissue homeostasis. interacts with the globular and the collagen-like regions of C1q on the surface of PS-positive cells at the early stages of apoptosis [20]. In turn, C1q binds to the phosphoserine CRT on the phagocyte surface group of PS through its globular domain, acting as a bridg- ing molecule [21]. CRT binds to PS with a ten-fold higher CRT on the surface of apoptotic or viable cells is able to affinity as compared to C1q binding, interfering with the interact with CD91 on