Stanniocalcin-1 and −2: The insulin-like growth factor system and beyond

The two stanniocalcins, STC1 and STC2, affect many cellular processes, yet the inhibition of PAPP-A and regulation of IGF signaling remain mechanistically the most well established, by structural, biochemical, genetic, and functional studies. Other functions, including calcium homeostasis, immune modulation, mitochondrial stress responses, integrins, DNA repair, and reproduction, are supported to varying degrees. Many questions are still unanswered, but due to the increasingly reported importance of both stanniocalcins in human physiology and diseases, it is important that future research disentangles the mechanisms and connections among these myriad functions. Fig. 1 Overview of reported interactors of STC1 (blue) and STC2 (yellow) categorized by topic area. Some topic areas might overlap with respect to stanniocalcin function. The figure was prepared using Inkscape v. 1.4.2 Overview of reported interactors of STC1 (blue) and STC2 (yellow) categorized by topic area. Some topic areas might overlap with respect to stanniocalcin function. The figure was prepared using Inkscape v. 1.4.2 Fig. 2 Simplified overview of the STC-PAPP-A-IGFBP-IGF axis. The liver secretes IGFs and IGFBPs into the circulation, where they form 1:1 complexes. When such complexes cross the endothelium into the microenvironment of target cells, bound, inactive IGF can be released from the IGFBPs through proteolytic cleavage by the pappalysins PAPP-A and PAPP-A2. The released, bioactive IGF activates IGFIR promoting downstream signaling. IGFs can also be secreted locally in tissues, where they function in an autocrine or paracrine fashion (lower left). STC1 and STC2 are able to inhibit the pappalysins, whereby IGF remains in the bound, bioinactive state (lower right). The molecular interactions have been recently reviewed ( 15 , 16 ). The figure was prepared using Inkscape v. 1.4.2 Simplified overview of the STC-PAPP-A-IGFBP-IGF axis. The liver secretes IGFs and IGFBPs into the circulation, where they form 1:1 complexes. When such complexes cross the endothelium into the microenvironment of target cells, bound, inactive IGF can be released from the IGFBPs through proteolytic cleavage by the pappalysins PAPP-A and PAPP-A2. The released, bioactive IGF activates IGFIR promoting downstream signaling. IGFs can also be secreted locally in tissues, where they function in an autocrine or paracrine fashion (lower left). STC1 and STC2 are able to inhibit the pappalysins, whereby IGF remains in the bound, bioinactive state (lower right). The molecular interactions have been recently reviewed ( 15 , 16 ). The figure was prepared using Inkscape v. 1.4.2 Fig. 3 Overview of the effect of the stanniocalcins on the Nrf2/HO-1/hemin pathway. In the Nrf2/HO-1/hemin pathway, Nrf2 upregulates HO-1 leading to the degradation of hemin to biliverdin. STC1 has been proposed to bind and protect hemin, while STC2 is proposed to bind both hemin and HO-1, potentially bringing them into close proximity. Both STC1 and STC2 have been reported to upregulate Nrf2, and STC2 was furthermore found to co-immunoprecipitate with Nrf2. The figure was prepared using Inkscape v. 1.4.2 Overview of the effect of the stanniocalcins on the Nrf2/HO-1/hemin pathway. In the Nrf2/HO-1/hemin pathway, Nrf2 upregulates HO-1 leading to the degradation of hemin to biliverdin. STC1 has been proposed to bind and protect hemin, while STC2 is proposed to bind both hemin and HO-1, potentially bringing them into close proximity. Both STC1 and STC2 have been reported to upregulate Nrf2, and STC2 was furthermore found to co-immunoprecipitate with Nrf2. The figure was prepared using Inkscape v. 1.4.2 Statements and Declarations.

Stanniocalcin (STC) is a glycoprotein first identified in the endocrine corpuscles of Stannius (CS) of teleost fish ( 1 ), where it functions as a hypocalcemic hormone maintaining serum calcium homeostasis in response to elevated calcium levels ( 1 , 2 ). Since the CS are only present in teleost fish and in holosteans, STC was thought to be a specific adaptation to aquatic environments, but almost ten years after its discovery, an STC homolog was identified in human tissues ( 3 , 4 ). A few years later, a second human STC paralog gene was identified ( 5 – 7 ), leading to the designation of STC1 and STC2. Structurally, both STC1 and STC2 form glycosylated, covalent homodimers. Their monomeric units contain 247 and 302 amino acids, respectively, including a signal peptide for secretion ( 3 – 6 ). Phylogenetic analysis has shown the presence of homologs of STC1 and STC2 during early vertebrate evolution, and STC homologs are predicted to appear as early as single-celled eukaryotes ( 8 , 9 ), suggesting deep evolutionary roots and fundamental cellular functions. Although first characterized in the context of calcium homeostasis, mammalian stanniocalcins have been implicated in a much wider range of biological processes, including ion transport, metabolic regulation, apoptotic and oxidative stress responses, growth and developmental processes, reproductive biology, and multiple aspects of tumor biology. Specifically, within the last 5–10 years, new functions for both stanniocalcins have been reported. This review will focus on reported functions of STC1 and STC2 that are substantiated mechanistically, i.e. in studies proposing direct molecular interactions (Fig.  1 ). We also emphasize overlaps between the functions and highlight potential gaps in the literature. We performed a PubMed database search for articles using the following search term “ STC1 OR STC-1 OR STC-2 OR STC2 OR Stanniocalcin ”. Only articles written in English were included and selected articles cited in the identified papers were also considered. STC1 and STC2 can potently inhibit the proteolytic activity of the two pappalysins, pregnancy-associated plasma protein-A (PAPP-A) and PAPP-A2. Structural, biochemical, genetic, and functional studies demonstrate that STC2 inhibits PAPP-A through a covalent interaction, thus blocking substrate entry. STC1 inhibits the pappalysins through high-affinity non-covalent interaction, but the binding mechanism is not as well studied. The inhibition of pappalysins by stanniocalcins leads to decreased insulin-like growth factor (IGF) activity, which manifests in mouse models and humans as a decrease in growth. In the decade following the discovery of mammalian stanniocalcins, Stc1 and Stc2 knockout (KO) mouse models were generated ( 10 , 11 ). Stc1 KO mice displayed no observed anatomical or histological abnormalities and, surprisingly, showed wild-type serum levels of calcium and phosphate after vitamin D 3 -induced hypercalcemia ( 11 ). To exclude the possibility that the STC1 loss was masked by STC2 compensation, the same authors produced double KO (DKO) mice, lacking both Stc1 and Stc2 . These mice also exhibited normal calcium and phosphate serum levels, suggesting that the stanniocalcins do not play a major role in mineral homeostasis in mice ( 10 ). Instead, the most notable phenotype was seen in the growth rates of the KO mice. Both DKO and Stc2 KO mice showed a faster postnatal growth rate than their wild-type littermates, and both body weight and body length were consistently increased (~ 15%) after weaning ( 10 ). Since no difference was found in total serum IGF levels in the Stc2 KO mice, it was concluded that the effect was independent of the IGF system ( 10 ). However, it was subsequently demonstrated that STC2 regulates the bioactivity of IGF-1 and IGF2, collectively referred to as IGF in this review, rather than total IGF levels, as STC2 was found to be an inhibitor of the growth-promoting pappalysins ( 12 – 14 ) (Fig.  2 ). The pappalysin family of metzincin metalloproteinases is constituted by two members: PAPP-A and PAPP-A2 ( 8 , 15 ). The pappalysins promote IGF signaling by releasing IGF from inhibitory IGF binding proteins (IGFBPs). The IGFBPs are a family of six proteins, of which PAPP-A cleaves IGFBP-2, -4 and −5 and PAPP-A2 cleaves IGFBP-3 and −5 ( 15 ). The IGFBPs bind both IGFs with high affinity, with IGFBP-3 being the most dominant carrier of IGF in the circulation ( 16 ). The released, bioactive IGF signals through the IGF receptor (IGFIR), activating the PI3K/Akt pathway, among other pathways, resulting in increased cellular proliferation, differentiation, and organismal growth ( 17 ). Thus, when the stanniocalcins inhibit the activity of the pappalysins, the amount of free, bioactive IGF is decreased, resulting in decreased signaling and growth ( 16 ). The molecular basis of this inhibition was clarified by recent structural studies showing the direct interaction between STC2 and PAPP-A. Two independent research groups solved the structure of the 2:2 heterotetramer PAPP-A: STC2 complex using cryo-electron microscopy (cryo-EM) ( 14 , 18 ). The structures revealed that STC2 adopts a homodimeric, all-helical conformation that is positioned at the upper central exosite of the PAPP-A dimer, where it sterically blocks IGFBP access without directly affecting the active site ( 14 ). Furthermore, Cys120 of STC2 forms a disulfide bond with Cys732 of PAPP-A ( 12 , 14 , 18 ), making the inhibition irreversible. There is no high-resolution structure of STC1, but low-resolution structural studies have revealed a homodimeric, compact, helical-dominated structure [ 19 ], indicating a similar overall structure to STC2. STC1 has also been shown to potently inhibit the proteolytic activity of the pappalysins ( 13 ). STC1 lacks an equivalent to STC2 Cys120 and instead inhibits through high-affinity interactions, with a dissociation constant ( K D ) of 75 pM against PAPP-A ( 13 ). Much less is known about the interaction between the stanniocalcins and PAPP-A2, but both STC1 and STC2 have been found to inhibit PAPP-A2 in vitro ( 13 ). Together, this shows that STC1 and STC2 indirectly inhibit the bioactivity of the IGFs by preventing release from IGFBPs. This was further substantiated in mice overexpressing either STC1 or STC2, which exhibit severe postnatal dwarfism with a 30–45% reduction in size ( 20 ). This phenotype is comparable to that of Pappa KO mice that exhibit 40% postnatal dwarfism ( 21 ) and Pappa2 KO mice which showed a size reduction of 10% for males and 30% for females ( 22 ). In humans, children expressing unstable or proteolytically inactive PAPP-A2 variants also exhibit decreased growth ( 23 – 25 ). Likewise, mice overexpressing STC2 Cys120Ala, which is unable to form a covalent complex with PAPP-A, showed normal growth ( 12 ). The normal growth of Stc1 KO mice might be explained by differences in expression patterns of STC1 and STC2. For example, STC2 has been shown to be constitutively secreted from human fibrosarcoma cells, while STC1 appeared to be sequestered internally and secreted after an external stimulus ( 26 ). Lastly, in humans, a large genome-wide association study identified an STC2 variant with a single amino acid substitution (Arg44Leu), which increased the height of carriers by 2.1 cm compared to non-carriers. This STC2 variant was found to have impaired PAPP-A complex formation ( 27 ), highlighting the connection between STC2 and the IGF system in humans ( 16 ). In summary, stanniocalcins affect the IGF system by inhibiting PAPP-A and PAPP-A2. These interactions, which lead to reduced growth, constitute the most well-founded molecular functions of STC1 and STC2, and they represent the only mechanism which is substantiated through biomolecular structures. In other biological systems, as reviewed in the following sections, molecular interactions with the STCs are generally supported less rigorously. Many findings are based solely on co-IPs with varying degrees of supporting control experiments, e.g. experiments designed to exclude indirect binding mediated by unknown interaction partners. All reported findings nevertheless warrant consideration, but further experimental analyses are needed to establish, for example, whether these interactions are direct, specific, and functionally relevant. STC1 and STC2 have long been associated with calcium homeostasis and regulation across vertebrates. Evidence from multiple systems suggests that the stanniocalcins affect cellular uptake and intracellular Ca 2+ signaling. In zebrafish, STC1 suppresses epithelial Ca 2+ uptake through IGF signaling. In mammalian cells, especially STC2 has been linked to suppression of store-operated Ca 2+ entry (SOCE), possibly through interaction with the endoplasmic reticulum (ER) Ca 2+ sensor stromal interaction molecule 1 (STIM1) as shown by co-immunoprecipitation (co-IP). The stanniocalcins play an important role in Ca 2+ homeostasis both dependent and independent of IGF signaling. The exact underlying mechanisms have remained elusive. The first biological action attributed to STC was its hypocalcemic action in teleost fish ( 1 , 2 ). It has been shown that activation of the calcium-sensing receptor (CaSR)-mediated signaling by high Ca 2+ concentrations results in increased expression of STC1 and STC2 ( 28 , 29 ). STC1 overexpression in zebrafish negatively regulates the number and functions of Ca 2+ -transporting epithelial cells (ionocytes) ( 30 ). Interestingly, recent work proposed that the effect of STC1a (one of the zebrafish orthologues of mammalian STC1) on ionocyte quiescence and proliferation is IGF dependent. It was found that the elevated ionocyte proliferation seen in stc1a null zebrafish was reversible through IGF1R inhibition, as well as by further knockdown of papp-aa or igfbp-5a ( 31 ). STC1 also inhibits epithelial Ca 2+ uptake by regulating the activity of the constitutively open calcium channels Trpv5 and Trpv6 in an IGF-dependent manner ( 32 , 33 ). Furthermore, it has recently been shown that in zebrafish larvae the STC1-PAPP-A-IGF-associated Ca 2+ regulatory pathway is involved in maintaining acoustic startle thresholds. As part of this mechanism, cadherin 16 functions as a negative regulator of STC1, resulting in increased Ca 2+ uptake ( 34 ). Together, these findings suggest that the mechanistic action of STC1 on repressing ionocytes and handling Ca 2+ uptake in zebrafish is manifested through its known role as a potent pappalysin inhibitor and hence IGF signaling, as detailed above. The role of STC1 in calcium homeostasis has recently been reviewed in more detail elsewhere ( 35 ). STC2 has been implicated in downregulation of SOCE. Intracellular Ca 2+ concentration depends on several different Ca 2+ transporters, including the ER Ca 2+ sensor STIM1. When the Ca 2+ concentration in the ER is low, STIM1 oligomerizes and binds to store-operated Ca 2+ channels (SOCs), such as Calcium release-activated calcium channel protein 1 (Orai1), in the plasma membrane, to allow for calcium entry into the ER ( 36 ). This process can be experimentally induced using Thapsigargin (Tg). Tg is an irreversible inhibitor of the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA), which depletes the ER of Ca 2+ and thus activates SOCE. Using this model, STC2 was found to be a negative regulator of SOCE ( 36 ). Murine embryonic fibroblasts (MEFs) from Stc2 null mice showed decreased resistance to Tg or H 2 O 2 treatment ( 36 ). Stc2 KO increased SOCE in response to Tg, an effect dependent on SOCs. Mechanistically, STC2 co-immunoprecipitated with STIM1 in a monkey kidney cell line, and interestingly this interaction did not depend on Ca 2+ concentration ( 36 ). STC2 also co-immunoprecipitated with STIM1 in MEFs, but only upon Tg or tunicamycin induced ER-stress. The translocation of STIM1 to the plasma membrane was not affected by STC2, so the way STC2 decreases SOCE is not clear, besides its possible interaction with STIM1 ( 36 ). A recent study confirmed the suppressive effect of STC2 on SOCE. Both in platelets from thrombotic patients and STC2-silenced platelet-progenitor cell lines, reduced STC2 correlated with higher SOCE. Furthermore, the authors also found co-IP of STC2 and STIM1 in human platelets ( 37 ). However, this study contrasts with an earlier study from the same group, where STC2 was proposed to regulate non-capacitative Ca 2+ influx ( 38 ). Although most mechanistic work on SOCE has focused on STC2, a later study has shown that rhSTC1 treatment of a human muscle cell line leads to suppression of STIM1 expression and SOCE, indicating that the two stanniocalcins may have similar roles in this regard ( 39 ). In summary, the stanniocalcins engage multiple pathways involved in calcium handling. The exact mechanisms are not clear and should be further studied, especially to disentangle how the calcium regulation depends on the roles of the stanniocalcins in the IGF system. Both stanniocalcins are strongly implicated in immune modulation, by influencing macrophage polarization, phagocytosis, and cytokine signaling. Specifically, STC1 has been found to reduce phagocytosis through trafficking the “eat-me” signal calreticulin (CRT) away from the cell surface. The interaction between STC1 and CRT might be regulated by an indicated interaction between STC1 and either A20 or Glycogen synthase kinase-3 beta (GSK3β). Although less mechanistically supported, STC1 can also affect macrophage polarization either by increasing the activity of the transcription factor Yes-associated protein (YAP), by interacting with p-21-activated kinase interacting exchange factor (βPIX), or by interacting with Glucose-regulated protein 94 (GRP94) or even the IGF2R. STC2 affects macrophage maturation by an interaction with histidine-rich glycoprotein (HRG), shown by several binding studies. Notably, the effects of the stanniocalcins on macrophage function can vary and can be either pro- or anti-inflammatory depending on the context, which reflects the complexity of the stanniocalcins and immune modulation. Macrophage functions, such as phagocytosis, are central to host immunity and tumor surveillance ( 40 ). Recent research has uncovered both stanniocalcins as modulators of the immune system, particularly by interfering with the polarization and phagocytic abilities of macrophages. The literature has shown several different mechanisms for this effect, and depending on the cellular context, the stanniocalcins can affect the immune system in opposite directions ( 41 – 45 ). Many of the reported immune functions of stanniocalcins involve macrophage polarization. Macrophages originate from monocytes that first mature into M0 macrophages that can then be polarized into a pro-inflammatory M1 state or anti-inflammatory M2 state, depending on signals in the microenvironment ( 40 ). In tumor microenvironments (TME), the tumor-associated macrophages (TAMs) have generally been polarized into the M2 type ( 40 ), thereby creating a safe zone for the tumor growth and immune evasion. STC1 is one of the factors in the TME that contributes to this imbalance. One of the most well-founded roles of STC1 in immune modulation is its interaction with CRT ( 46 – 49 ). Normally residing in the ER, CRT functions as an “eat-me” signal when presented on the cell surface ( 50 ). By interacting with antigen-presenting cells, such as macrophages, CRT can increase phagocytosis and thus lead to T cell activation ( 46 ). STC1 reduces phagocytosis by affecting CRT localization. In murine tumor models from three different cancer types, immunofluorescence, cell fractionation, and co-IP showed that STC1 traffics CRT from the cell surface to the mitochondria ( 46 ), while co-culture assays confirmed that STC1 from tumor cells decreases phagocytosis and T cell activation. At the same time, this effect was lost if the tumor cells lacked CRT ( 46 ). A subsequent study in three cancer cell lines confirmed this mechanism ( 47 ), where STC1 again interacted with CRT and promoted CRT trafficking to the mitochondria, in a manner dependent on A20, an anti-inflammatory protein. Co-IP showed an association between A20 and STC1, which stabilized STC1 protein levels. Co-IP furthermore showed an interaction between GSK3β and STC1, which is reduced by A20, proposing a model where A20 stabilizes STC1 by inhibiting GSK3β-phosphorylation and subsequent degradation of STC1 ( 47 ). The interaction between STC1 and GSK3β is also interesting, since GSK3β is known to regulate the NF-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/Hemin pathway ( 51 ), which both stanniocalcins are involved in, as described later in this review. Finally, a recent study has shown the same effect of STC1-mediated sequestering of CRT to the mitochondria in a human breast cancer cell line ( 48 ). Together, these studies establish a mechanism where STC1 can reduce phagocytosis by removing the “eat-me” signal CRT from the cell surface and traffic it instead to the mitochondria. Besides affecting phagocytosis by trafficking CRT, STC1 can affect polarization and maturation of macrophages. In melanoma cancer cells, STC1 increased invasion, migration, and protein expression of C-C Motif chemokine 2 (CCL2), Programmed cell death 1 ligand 1 (PD-L1), and epithelial-mesenchymal transition (EMT) markers, while increasing M2 polarization and vascular endothelial growth factor A (VEGFA) secretion from macrophages ( 41 ). Since CCL2 is a target gene of the transcription factor YAP, the relationship between YAP and STC1 was investigated. STC1 was shown to activate YAP signaling, and YAP inhibition could remove the effect of STC1 on not only CCL2 expression, but also migration, M2 polarization, and expression of PD-L1, and EMT markers. No interaction was found between STC1 and YAP. Instead, both proteins co-immunoprecipitated with βPIX, both interacting with the same KER domain of βPIX. The authors propose that the effect of STC1 on macrophages results from the competitive binding of STC1 to βPIX which releases YAP and allows for its activation and nuclear translocation, supported by the finding that the effect of STC1 on YAP activation is removed if βPIX is silenced ( 41 ). In contrast, one study found that STC1 inhibits macrophage maturation through the secreted protein GRP94 ( 44 ). Tumor-associated fibroblasts secrete STC1, and STC1 pull-down assays coupled with mass spectrometry indicated an interaction between secreted STC1 and GRP94, which interacts with the class A1 scavenger receptor (SR-A) on immature macrophages to induce maturation ( 44 ). However, STC1 inhibited the interaction between GRP94 and SR-A, leading the authors to propose a model in which STC1 secreted by tumor-associated fibroblasts sequesters GRP94, thereby inhibiting SR-A receptor activation and macrophage maturation ( 44 ). Furthermore, human lung adenocarcinoma with high STC1 expression has decreased polarization from M0 to M2 ( 44 ). This contrasts with several studies showing that STC1 induces polarization of macrophages from M0 to M2 ( 41 – 43 , 52 , 53 ). This adds to the emerging pattern of STC1 as having complex and competing roles, depending on cellular context or tissue type. Another way STC1 potentially regulates phagocytosis is via the IGF2 receptor (IGF2R). Using TriCEPS ligand-receptor capture and surface plasmon resonance, a high-affinity interaction between IGF2R and STC1 was found ( 54 ). The authors then showed that hSTC1 treatment of THP-1 cells reduces interleukin-1β (IL-1β) secretion, but this effect disappears if IGF2R is silenced, thus supporting the role of STC1 as a negative modulator of macrophage activity ( 54 ). However, since IGF2 interacts with the IGF2R to affect IL-1β ( 55 ), the interaction between STC1 and IGF2R might hypothetically be connected to STC1’s modulation of IGF signaling through PAPP-A, rather than being a separate function. Evidence has emerged that STC2 contributes to immune regulation through complex formation with HRG, as shown by co-IP ( 56 , 57 ). HRG promotes macrophage phagocytosis and favors M1 over M2 polarization, in competition with the immune-suppressive nature of STC2. STC2 treatment of U937 monocytes reduced the proportion of CD14 + cells, thereby inhibiting macrophage maturation, an effect reversed by co-treatment with HRG ( 57 ). The same laboratory co-immunoprecipitated purified STC2 and HRG and quantified the binding affinity using Quartz Crystal Microbalance (QCM) technology, finding a K D of 55 nM. QCM analysis also showed that both proteins can bind to differentiated U937 cells ( 56 ). The same study showed that HRG significantly increased phagocytosis, but co-treatment with STC2 reversed this effect. Together, these two studies show that the regulatory effects of STC2 and HRG are dependent on cellular context, but both underline the role of STC2 in suppressing immune activation. Collectively, STC1 and STC2 can be portrayed as active components of immune microenvironments. Through CRT sequestration, GRP94 binding, IGF2R engagement, YAP activation, and HRG regulation, the mechanisms are complex and broad. Compared with the structural basis and mechanistic clarity of IGF regulation, these mechanisms are less well established. Clarifying these mechanisms could help explain the dual roles of STC1 and STC2 in modulating the immune system in opposite directions, offering critical insights into how the TME affects tumor immune evasion. Mitochondrial regulation and control of oxidative stress represent some of the most cited but also most complex functions of STC1 and STC2. Both stanniocalcins act as modulators of mitochondrial function, cellular energy metabolism, and antioxidant defense. STC1 can be trafficked to the mitochondria through a megalin-dependent pathway. Here, STC1 modulates energy metabolism and oxidative stress, possibly through its interactions with Sirtuin 3 (SIRT3), ATP, and hemin. STC2 also plays a role, especially in antioxidant defense and is proposed to interact with hemin, HO-1, and Nrf2. Here, the interaction between STC2 and HO-1 is the best supported by yeast-2-hybrid (Y2H) and co-IPs. Although the stanniocalcins both have roles that converge on maintaining mitochondrial integrity and redox balance, they do so through different mechanisms that are not all completely understood. STC1 has been found to increase both mitochondrial respiration and glycolytic function in mouse embryonal fibroblasts (MEFs), an effect depending on megalin-mediated trafficking of STC1 ( 58 ). Secreted STC1 is proposed to be internalized by binding to megalin on the cell surface as shown by co-IP ( 58 , 59 ) and this interaction was disrupted by mutating leucines in the signal peptide of either protein ( 58 ). The trafficking from the cell surface to the mitochondria was dependent on Rab32, which is involved in vesicular transport between the ER/Golgi and the mitochondria ( 59 ). STC1 and megalin also co-immunoprecipitated with SIRT3 ( 59 ). Since SIRT3 is involved in antioxidant defense ( 60 ), this positions SIRT3 as a potential downstream effector of the antioxidant role of STC1. These findings establish a route for secreted STC1 to enter the cell and subsequently the mitochondria, thereby directly affecting mitochondrial function. Once in the mitochondria, STC1 can affect several aspects of mitochondrial physiology, including respiration and redox state. An early study showed that STC1 increased state 3 oxygen consumption and decreased the ADP:O ratio, resulting in a higher respiration rate but with lower efficiency ( 61 ). STC1 could bind to mitochondria and to the membrane of kidney epithelial cell lines, but this binding was reduced when treated with nucleotides, especially ATP ( 61 ). Furthermore, a consensus protein kinase ATP-binding cassette signature with no catalytic domain in the STC1 sequence was identified, supporting the idea STC1 might be modulated by ATP binding ( 61 ). An interaction between STC1 and ATP was indeed shown using co-IP of STC1 and radiolabeled ATP ( 61 ), indicating that the effect of STC1 on mitochondrial function happens at least partially through binding to the mitochondria and that this effect is modulated by the amount of ATP present. This supports a complex, context-dependent relationship between STC1 and cellular ATP. The direction of STC1’s effect on ATP production seems ambiguous ( 42 , 62 – 64 ). One possible mechanism for how STC1 affects ATP production is through the regulation of uncoupling proteins (UCPs). Most studies agree that STC1 activates the AMP-activated protein kinase (AMPK) pathway, which leads to both upregulation of UCP2 ( 60 , 62 , 65 – 69 ) and SIRT3 ( 60 , 66 , 70 ). In contrast, a few studies found that STC1 did not affect UCP2 levels, but instead increased UCP3 levels and that the protection by STC1 against reactive oxygen species (ROS) generation was dependent on UCP3 ( 71 , 72 ). The STC1/AMPK/UCP2/SIRT3 pathway contributes to protection against ROS generation ( 62 ), but it also leads to lower efficiency in ATP production. In contrast, a study that did not examine UCP levels found that STC1 increases mitochondrial coupling efficiency in mouse lung endothelial cells (MLECs) ( 73 ). Since the mechanism by which STC1 affects the AMPK pathway is unclear, it is challenging to determine how STC1 can influence ATP production in both directions. However, the activation and phosphorylation of AMPK are tightly regulated depending on cellular energy levels, calcium levels, and oxidative stress ( 74 ). A recent study has found a possible connection between the STC1 interaction with CRT and the activation of AMPK ( 49 ), as described below. Beyond AMPK-mediated effects on ROS, STC1 and STC2 have been implicated in cellular antioxidant defense through a pathway centered on the transcription factor Nrf2. Nrf2 promotes defense against oxidative stress, partially through its upregulation of HO-1, which degrades hemin to biliverdin, leading to anti-inflammatory and antioxidant effects ( 75 ). The stanniocalcins affect all three components of the Nrf2/HO-1/Hemin pathway ( 63 , 76 – 80 ) (Fig.  3 ). Both STC1 and STC2 contain a heme regulatory motif (HRM) with a cysteine residue that is proposed to facilitate the binding of the stanniocalcins to hemin ( 76 , 77 ). Mutating these two reported cysteine residues, Cys114 for STC1 and Cys125 for STC2, into alanines results in the loss of heme binding and therefore it was proposed that these cysteines are required for hemin binding ( 76 , 77 ). However, in the PAPP-A:STC2 complex, this cysteine is already engaged in intramolecular disulfide bridges ( 12 , 14 ). Therefore, the intervention of mutating these cysteines might have led to protein misfolding that could explain the observed changes in potential binding patterns. Functionally, STC1 protects hemin from H 2 O 2 -mediated decay in vitro ( 77 ). Interestingly, STC2, but not STC1, was found to interact with HO-1 ( 76 ). Y2H and co-IPs showed that the interaction depends on amino acids 181–200 of STC2 ( 76 ). Since this region is distinct from the hemin-binding region, the authors propose that STC2 may bind hemin and HO-1 simultaneously, thereby delivering hemin to HO-1 as a substrate for degradation. The interaction between STC2 and HO-1 has also been found by co-IP in mesenchymal stromal cells (MSCs) ( 78 ). STC2 was shown to increase the enzymatic activity of HO-1, and this effect was also dependent on the STC2 amino acids 181–200 ( 78 ), further supporting that residues 181–200 of STC2 form the interface of the STC2:HO-1 interaction. An anti-inflammatory effect of STC2 was also dependent on this region, possibly implicating the Nrf2 pathway as a mechanism for the anti-inflammatory activity of STC2 ( 78 ). Both stanniocalcins influence Nrf2, which is upstream of HO-1. STC2 co-immunoprecipitated with Nrf2 in both the cytoplasm and the nucleus ( 80 ), and upregulated both Nrf2 and HO-1 mRNA levels ( 80 ). STC1 was shown to have a protective effect on cells that are mitochondrially challenged by treatment with iohexol. This was through the Nrf2 pathway by increasing the levels of both Nrf2 and HO-1 ( 79 ). Together these findings describe complementary roles of STC1 and STC2, where STC1 stabilizes hemin ( 77 ) and STC2 facilitates hemin turnover ( 76 ), with both stanniocalcins contributing to the Nrf2/HO-1/Hemin pathway ( 76 – 80 ). Interestingly, a recent study has found a possible connection between several of the proposed functions of STC1 resulting in activation of the Nrf2/HO-1/Hemin pathway ( 49 ). STC1 interaction with CRT led to the activation of the calmodulin-dependent protein kinase kinase 2 (CAMKK2), which in turn activated the AMPK-GSK3β-Nrf2-HO-1 axis, functionally leading to reduced oxidative stress and apoptosis ( 49 ). The authors proposed that the STC1-mediated AMPK activation is dependent on the interaction between STC1 and CRT, which regulates calmodulin levels and activates CAMKK2, a known activator of AMPK ( 49 ). The study highlights the importance of integrating and connecting the complex roles of the stanniocalcins. Collectively, these studies elucidate the multiple mechanisms by which the stanniocalcins regulate cellular energy metabolism and redox state, often converging on mitochondrial function. However, it remains unclear how and when STC1 activates the AMPK pathway, how it influences mitochondrial uncoupling, and whether the stanniocalcins act cooperatively or counteract each other in regulating the Nrf2 pathway. Uncovering these mechanisms will be important to understand how the stanniocalcins regulate metabolism and cellular stress defense. Recent evidence has linked STC1 to modulation of Notch signaling. Interaction between STC1 and Notch is suggested by co-IPs. However, the nature of the interaction remains unclear. The Notch receptor family is highly conserved across metazoans, in which Notch signaling is involved in organ development, cell fate, and tissue homeostasis ( 81 ). Notch signaling is furthermore implicated in several cancers, where, depending on the context, it can either promote or inhibit cancer development ( 81 , 82 ). Canonical Notch signaling is induced through interaction between the Notch receptor and ligands on a juxtaposed cell. This results in proteolytic cleavage of the Notch receptor to form the Notch intracellular domain (NICD), which regulates the transcription of target genes ( 81 , 82 ). In addition to canonical signaling, there are several noncanonical pathways, some of which are dependent on soluble, extracellular ligands ( 83 ). Of the known mammalian noncanonical ligands, no consensus binding site has been identified, but they all seem to bind the extracellular domain (ECD) of Notch, with several reported to bind the tandemly arrayed epidermal growth factor (EGF)-like repeats ( 83 , 84 ). Recently, STC1 has been reported to affect cell proliferation and stemness by modulating Notch1 signaling in two different cancer types ( 85 , 86 ) and psoriasis ( 87 ). Here, STC1 was found to induce Notch1 cleavage in glioblastoma, hepatocarcinoma, and primary and secondary keratinocytes, and subsequent STC1 silencing was sufficient to reverse this effect ( 85 – 87 ). In all three studies, this modulation of Notch1 signaling was reported to occur through an association between STC1 and Notch1, as evidenced by co-IP. However, results on interaction sites were conflicting, with one report suggesting the EGF repeats 1–24 of the Notch1 ECD to be responsible ( 86 ), while another suggested an interaction with the NICD ( 87 ). In summary, STC1 may function as an agonistic, secreted, noncanonical Notch1 ligand that acts in an autocrine/paracrine manner to stimulate Notch1 signaling either in the TME or in inflamed keratinocytes. However, it is also possible that STC1 functions in a similar way to the noncanonical Notch3 ligand thrombospondin-2 (TSP2), in which TSP2 is insufficient to promote Notch3 activation, but instead enhances the complex formation between Notch3 and Jagged1 ( 88 ). Therefore, characterization of how STC1 interacts with Notch1 and whether the stimulating effect of this interaction depends on canonical ligands needs further study. The stanniocalcins can influence cell signaling through engaging integrins. While co-IPs and functional studies suggest that STC1 interacts with integrin beta-6 (ITGB6) and STC2 interacts with integrin beta-2 (ITGB2), both stanniocalcins use integrins to increase migration and metabolism. Integrins are transmembrane dimeric proteins whose extracellular domain interacts with the extracellular matrix while their cytosolic domain interacts with signaling components ( 89 ). When the integrins are engaged, they recruit and activate Focal adhesion kinase 1 (FAK), leading to downstream activation of the PI3K/AKT pathway, ultimately affecting cell survival, proliferation, EMT, and metabolism ( 89 ). Consistent with this signaling framework, STC1 has been linked to integrin-dependent activation of the PI3K/AKT pathway, with two reports linking this to interaction with integrin subunit ITGB6 ( 90 , 91 ). STC1 was reported to activate the PI3K/AKT pathway leading to increased cell survival, migration, and EMT in two gastric cancer cell lines, possibly via an association with the integrin subunit ITGB6, as evidenced by co-IP ( 90 ). STC1 has also been shown to enhance both metastasis and lipid metabolism in ovarian cancer cell lines ( 91 ). Specifically, silencing of STC1 reduced UCP1 protein levels in both cell lines and mouse tumors. STC1 co-immunoprecipitated with ITGB6 in an ovarian cancer cell line ( 91 ) and although the mechanism was not clear, both STC1 and ITGB6 converged on activation of the PI3K/AKT pathway ( 91 ). This indicates that STC1, through interaction with ITGB6, can influence integrin-mediated signaling, specifically leading to alterations in energy metabolism. However, it is important to note that the PI3K/AKT pathway is not specific to integrin signaling but is also activated by IGF1R signaling ( 17 ). Since STC1 downregulates signaling of the IGF system by inhibiting the pappalysins, it is paradoxical that STC1 at the same time activates downstream components of the IGF system by interacting with ITGB6. Further studies are needed to delineate to what extent the effects of STC1 on the PI3K/AKT pathway arise through separate or shared mechanisms. STC2 promoted EMT, migration, invasion, and glycolysis in nasopharyngeal cancer cell lines by engaging the integrin/AKT/SOX6 signaling cascade ( 92 ). Mechanistically, STC2 co-immunoprecipitated with ITGB2 in a nasopharyngeal cancer cell line, and ITGB2 overexpression rescued the effects of STC2 silencing on migration, invasion, and SOX6 protein levels ( 92 ). This finding was supported by a recent study on glioma cell lines, where STC2 similarly enhanced proliferation, invasion, migration, EMT, and glycolysis. Suppression of these processes following STC2 silencing was completely or partially rescued by ITGB2 overexpression ( 93 ). Overall, STC2 was shown to interact with ITGB2 leading to multiple effects, including enhanced glycolysis in both nasopharyngeal cancer cells and glioma cells ( 92 , 93 ). This positions STC2 as a metabolic modulator, shifting energy production toward glycolysis. STC1 and STC2 both appear to interact with integrins, thereby influencing signaling and energy metabolism. However, further studies are needed to determine how the interaction between the stanniocalcins and the integrins affects integrin signaling activity. The three studies examined cancer cells, but given the literature supports the notion that STC1 has dual roles depending on cellular context, it would be interesting to determine whether the STC1:integrin interaction depends on the TME or on stress signals. An important part of this puzzle is to determine when the stanniocalcins act through integrin signaling versus modulation of the IGF system. Both stanniocalcins play roles in modulating DNA damage repair (DDR) regulation. Notably, STC1 exhibits context-dependent effects on DDR. Evidence from co-IP suggests an interaction between STC1 and the breast cancer type 1 susceptibility protein (BRCA1), leading to a protective effect on DDR. In contrast, several co-IPs suggest an interaction with poly(ADP-ribose) polymerase 1 (PARP1), which skews the role of STC1 towards a damaging effect. STC2 upregulates DDR by interacting with a DDR complex, consisting of protein methyltransferase 5 (PRMT5), chloride nucleotide-sensitive channel 1 A (CLNS1A), and WD repeat domain 77 protein (WDR77). STC1 might function as a small ubiquitin-like modifier 1 (SUMO-1) E3 ligase by interacting with SUMO-1, as shown by co-IP and Y2H. This suggests a mechanism by which the stanniocalcins affect the stability of DDR-related proteins, but further studies are needed. DNA double-stranded breaks (DSBs) are one of the most cytotoxic forms of DNA damage and thus efficient DDR is essential for cell survival ( 94 ). Mammalian cells mostly use nonhomologous end-joining (NHEJ) and homologous recombination (HR) to repair DSBs, both pathways being tightly regulated by numerous co-factors ( 94 ). DSB formation triggers phosphorylation of histone H2AX, forming γH2AX foci which can be measured and thus used as a DSB quantifier ( 94 ). The literature shows that stanniocalcins participate in both the formation of DSBs and the repair, influencing HR and NHEJ ( 95 – 97 ). STC1 has been shown to affect DDR in both directions. On the protective side, STC1 has been found to decrease DSBs after temozolomide treatment ( 98 ), and to enhance HR-mediated DNA repair and cell survival following irradiation ( 95 ). Mechanistically, STC1 increased the efficiency of DDR in breast cancer cell lines through BRCA1 recruitment to the DNA damage, thus facilitating HR ( 95 ). STC1 and BRCA1 co-immunoprecipitated, increasing after irradiation ( 95 ). However, the precise mechanism by which STC1 promotes BRCA1 localization to DSBs remains unclear. Future studies should clarify whether STC1 actively recruits BRCA1 to the DNA damage site or regulates BRCA1 stability. In contrast, STC1 is also linked to the accumulation of DNA damage under oxidative stress ( 96 ). In H 2 O 2 -treated colon-derived cells, STC1 increased the number of γH2AX foci, indicating enhanced DSB formation ( 96 ). This suggests a shift in the role of STC1 from protective to damaging, through a mechanism in which STC1 promotes PARP1-dependent cell death (parthanatos). Parthanatos is a pathway triggered by cellular damage, which starts with activation of PARP1 and ends with apoptosis-inducing factor (AIF) translocating to the nucleus with a DNA nuclease, thus leading to DNA cleavage and ultimately cell death ( 99 ). STC1 and PARP1 co-immunoprecipitated in colon cells and in a non-cellular setup using purified proteins, an interaction requiring the third zinc finger of PARP1 ( 96 ). STC1 stabilized PARP1 by inhibiting its ubiquitination, thus prolonging its activity ( 96 ). PARP1 supports DDR under limited damage but triggers parthanatos upon overactivation ( 96 ), so STC1 may act as a modulator of this threshold by affecting the stability of PARP1. Besides affecting the stability of PARP1, STC1 has been implicated in protein stability and post-translational modifications through SUMOylation ( 100 ). Y2H screening identified SUMO-1 as a possible interaction partner. STC1 co-immunoprecipitated with SUMO-1 in HEK293 cells and increased SUMOylation of Ran GTPase-activating protein 1 (RanGAP-1), suggesting that STC1 can function as an E3 SUMO-1 ligase ( 100 ), thereby highlighting a possible pattern of STC1 affecting the stability of target proteins related to DDR or otherwise. STC2 has also been implicated in the regulation of DDR. In esophageal cancer cells, STC2 increased cell survival after irradiation and decreased DSBs, measured by γH2AX foci ( 97 ). Mechanistically, STC2 co-immunoprecipitated with PRMT5, CLNS1A, and WDR77, a complex involved in DDR. Furthermore, STC2 was suggested to activate PRMT5 and thereby lead to upregulation of DDR proteins. However, the effects of STC2 on the investigated DDR proteins were inconclusive, especially for the NHEJ proteins ( 97 ). Even though the exact mechanism is not clear, STC2 had a significant protective effect against DNA damage, supported by a recent study, which correlates STC2 levels with radioresistance in lung cancer ( 101 ). Together, these findings illustrate that the two stanniocalcins can play divergent roles in DDR. The dual role of STC1 shows how different cellular cues and stress signals can shape its direction of action. Here, different cell types and stress conditions are sufficient to turn STC1 from a protective factor that increases DDR to a harmful factor that increases DNA damage. STC2 was associated with enhanced repair after irradiation, but further studies should investigate how STC2 affects DDR after oxidative stress to determine whether STC2 is a consistent protective factor. The stanniocalcins, especially STC1, have been repeatedly linked to reproductive physiology. This association does not stem from well-defined molecular interactions, but rather from consistent molecular observations and strong clinical correlations between altered stanniocalcin levels and pathologies in reproductive tissues. The only identified role based on association is between STC2 and the human testes protein Ran-binding protein microtubule-organizing center (RanBPM) and the androgen receptor (AR), as suggested by co-IP. When STC2 interacts with RanBPM, it increases RanBPM-mediated activation of the AR. However, even though an association has been shown, the specific effect of this activation on androgen-sensitive cells remains unknown. Dysregulation of STC1 or STC2 levels has been associated with complications of reproductive tissues including ovarian cancer, endometriosis, pre-eclampsia, and polyendocrine metabolic ovarian syndrome (PMOS) ( 102 , 103 ). Besides disease associations, one clue to the role of STC1 in reproductive tissues is the rise of circulating STC1 during pregnancy. STC1 rises in the maternal serum during pregnancy, in both humans and mice ( 103 ). The origin of the extra STC1 is not known, but in mice the increase in serum STC1 coincides with an increase of ovarian STC1 mRNA levels ( 104 ), suggesting the ovaries as a possible source for the increase in circulating STC1. Both stanniocalcins can be seen in human ovarian tissue by immunohistochemical staining, co-localizing with PAPP-A ( 105 ), providing the spatial proximity for the stanniocalcins to inhibit PAPP-A, as discussed above in the “The IGF system” section. PAPP-A levels also rise during pregnancy, with peak levels in the 3rd trimester ( 16 ). The molar PAPP-A levels in pregnancy are higher compared to STC1, and the majority of PAPP-A is bound to and inhibited by the placentally derived proform of eosinophil major basic protein (proMBP) ( 15 ). Beyond the presence of conventional 50 kDa STC1 ( 2 ), ovarian tissue apparently also contains unique high molecular weight forms, collectively referred to as Big STC1. Several studies from the same research group ( 106 – 111 ) found Big STC1 produced in the ovary, as variants of approximately 85, 112, and 135 kDa ( 110 ). Big STC1 was found to be produced in theca cells of rat and bovine ovaries ( 110 ) and to bind to luteal cells of bovine ovaries, ultimately leading to a reduction in progesterone secretion from these luteal cells ( 109 ). Even though 50 kDa STC1 could displace Big STC1 binding on bovine luteal cells, it did not have the same effect on progesterone secretion, suggesting a different functionality among the Big STC1 variants. Besides ovarian expression, both STC1 production and binding sites have been found in rat mammary glands, and these are affected by pregnancy and lactation stage ( 111 ). The same authors also detected Big STC1 from the nucleus of rat mammary glands, which they hypothesized originated from ovarian STC1 production ( 111 ). Overall, this points to STC1 having an active role in the physiological adaptations during pregnancy as well as in lactational function. Compared to females, the role of stanniocalcins in male reproduction is less well studied. However, one study has found an association between STC2 and two proteins involved in male reproduction, RanBPM and AR using Y2H analysis with a human testis cDNA library ( 112 ). RanBPM can increase the transcriptional activity of nuclear receptors, thereby modulating the expression of their target genes. STC2 co-immunoprecipitated with RanBPM and the specific nuclear receptor, AR, especially if co-treated with the AR ligand. Thus, STC2 could boost the ability of RanBPM to regulate transcriptional activity of the liganded nuclear receptor AR ( 112 ). This links STC2 to androgen-responsive pathways in the testis, but the exact physiological relevance is still unknown. No relevant well-founded mechanism has been established to explain how STC1 or STC2 affects reproductive physiology. However, the combination of altered expression levels and tissue-specific variants, binding sites and interactions indicates that the stanniocalcins modulate key physiological states in reproduction. Clarifying how STC1 and STC2 influence both reproductive health and the development and success of pregnancy requires further investigation.