Stanniocalcins (Stcs) are evolutionarily conserved glycoproteins. The first Stc protein was discovered from the Corpuscles of Stannius (CS), an endocrine organ unique to bony fish (1, 2). Surgical removal of CS resulted in elevated blood calcium levels and the appearance of kidney stones (3–5). Injection of CS extracts or purified Stc1 reversed these effects (6). In cultured rainbow trout CSs, secretion of Stc1 was found to be regulated by external Ca²⁺ levels ([Ca²⁺]) (6). High [Ca²⁺] increased Stc1 secretion via the calcium sensing receptor (7). In vivo, zebrafish embryos raised in high [Ca²⁺] media showed elevated stc1 mRNA levels (8, 9). Morpholino-based knockdown of Stc1a increased Ca²⁺ uptake and forced expression of Stc1a decreased Ca²⁺ uptake (10, 11). These and other findings have led to the notion that Stc1 is a hypocalcemic hormone in fish (2, 4, 6).

For several decades, Stc1 was considered a fish-specific hormone and even once called teleocalcin (2). Recent advances in genomics, however, have revealed that two STC genes are present in humans and other mammals. Human STC1 shares 61% sequence identity with fish Stc1 (12). In addition to STC1, there is a related protein (STC2), which shares ~30% identity in amino acid sequence with STC1 and contains a histidine cluster in the C-terminal region (2). Subsequent studies show that many teleost fish including zebrafish have 4 distinct stc genes, including stc1a, stc1b, stc2a, and stc2b (13), consistent with the notion that many teleost fish genomes underwent an additional round of genome-wide duplication (14). Published results suggest that mammalian STCs regulate somatic growth by inhibiting the insulin-like growth factor (IGF) signaling locally (15–17). IGFs act by binding to the IGF1 receptor and activating the downstream signaling cascades, including the PI3K-AKT-mTOR pathway and the RAS/RAF-MAP kinase pathway (18). In extracellular environments, IGFs are found in complexes with six types of IGF binding proteins (IGFBPs). These IGFBPs bind to IGF with an equal or greater affinity than the IGF1 receptor and therefore regulates IGF availability and biological activity (19). An important regulatory mechanism of the IGF signaling is proteolytic degradation of IGFBPs (16). Two structurally related metalloproteinases, pregnancy-associated plasma protein-a (PAPP-A) and PAPP-A2, have been shown to cleave IGFBPs and release IGFs from the IGFBP-IGF complex for IGF1 receptor binding (20). In vivo and biochemical studies suggest that human STC1 and STC2 function as potent inhibitors of PAPP-A and PAPP-A2 (21–23).

Recent genetic studies in zebrafish suggest that Stc1a is essential for life (24). stc1a ^-/- zebrafish developed cardiac edema around 4-5 days post fertilization (dpf). This was followed by whole body swelling and premature death (24). In zebrafish, calcium uptake is mainly carried out by Na+/H⁺-ATPase-rich (NaR) cells, one of the five types of ionocytes (25). stc1a^-/- mutant larvae had significantly more NaR cells due to elevated NaR cell proliferation (24). Mechanistic analysis results show that Stc1a suppresses local IGF signaling by inhibiting Papp-aa mediated degradation of IGF binding protein 5a (Igfbp5a) in NaR cells (24, 26). A loss of Stc1a liberates IGFs from the Igfbp5a/IGF complex and increases bioavailable IGFs for IGF1 receptor binding (24, 26, 27). Addition of fish IGF-1 in excess was sufficient to increase NaR cell proliferation (26, 28). These findings suggest that the Stc1a-Papp-aa-Igfbp5a-IGF axis regulates NaR cell number and density.

A hallmark of NaR cells is the expression of Trpv6 (previously known as epithelial calcium channel or ECaC) (10, 29). Trpv6 is a constitutively open channel and it mediates continuous Ca²⁺ influx and maintains high cytoplasmic [Ca²⁺] levels (29). We have previously shown that genetic deletion of trpv6 not only reduces calcium influx but also increases NaR cell proliferation (29). While trpv6^-/- fish suffered from calcium deficiency and died prematurely, stc1a^-/- fish had elevated body calcium levels but also died prematurely (24, 29). The relationship between Stc1a, Trpv6, and IGF signaling in regulating NaR cell proliferation and calcium uptake is unclear. In the current study, we provide evidence that both Stc1a and Trpv6 inhibits NaR cell proliferation by suppressing IGF signaling. Genetic deletion of Stc1a increases trpv6 mRNA levels and results in abnormal calcium deposits in the yolk sac and kidney stones. These phenotypes were rescued by inhibiting Trpv6 channel activity and by double knockout of Trpv6. Additional evidence suggests a crosstalk between Trpv6-mediated calcium signaling and IGF signaling in NaR cells and they work together to maintain calcium homeostasis and organismal survival.

Stc1a is synthesized and secreted from CS (10) ( Figure 1A ). As previously reported, genetic deletion of Stc1a resulted in a significant increase in NaR cells (24) ( Figures 1B, C ). Whether this action of Stc1a is specific to NaR cells was not clear. In this study, we determined the number of H⁺-ATPase-rich (HR) cells and Na⁺/Cl^_ cotransporter (NCC) cells, two other ionocyte types responsible for Na⁺ uptake and Cl^- uptake (25). No significant difference was detected in either HR cells or NCC cells ( Figures 1D–G ) between stc1a ^-/- larvae and their siblings, suggesting the action of Stc1a is specific to NaR cells. This result is consistent with previous studies showing that Igfbp5a is specifically expressed in NaR cells, but not in other ionocyte types (30–32).

To test whether Trpv6 is involved in the increased NaR cell proliferation observed in stc1a ^-/- mutant fish, we measured trpv6 mRNA levels by qRT-PCR in Tg(igfbp5a:GFP) fish. In Tg(igfbp5a:GFP) fish, NaR cells are genetically labeled by GFP expression (32), allowing quantification of NaR cells in live larvae. Compared to the siblings, stc1a^-/- fish had significantly greater levels of trpv6 mRNA ( Figure 2A ). To ascertain that the increased trpv6 mRNA levels are not a result of increased NaR cell number in stc1a^-/- fish (24), GFP-positive NaR cells were quantified and used to normalize trpv6 mRNA levels. The trpv6 mRNA levels/NaR cell in stc1a ^-/- were also significantly greater than the siblings ( Figure 2C ). Our finding is consistent with in vitro studies reporting that si/shRNA-mediated knockdown of STC1 increases TRPV6 protein levels in human CaCo2, Hela, and Caski cells (33, 34). Next, we measured stc1a mRNA levels in trpv6^-/- fish and siblings. As shown in Figure 2B , stc1a mRNA levels were significantly lower in trpv6^-/- mutant fish, suggesting that Stc1a and Trpv6 are interconnected.

Previous studies have shown that loss of Stc1a increases IGF-Akt-Tor signaling in NaR cells (24). Does the elevated IGF-Akt-Tor signaling play any role in the increase of trpv6 mRNA expression in stc1a^-/- fish? This idea was tested by treating stc1a ^-/- fish and siblings with BMS-754807, an IGF1-R inhibitor (31). As shown in Figures 2C, D , BMS-754807 reduced trpv6 mRNA levels to the sibling levels, suggesting that loss of Stc1a increases Trpv6 expression via an IGF signaling-dependent mechanism. Recently, we have discovered that serum- and glucocorticoid-regulated kinase 1 (Sgk1) acts downstream in the IGF-Akt-Tor signaling pathway in NaR cells (35, 36). Studies in culture mammalian cells suggest that SGK1 up-regulates the expression of several ion channels and transporters, including the epithelial Ca²⁺ channels TRPV5 and TRPV6 (37). SGK1 influences transcription factors such as NF-κB, p53, CREB, AP-1 and FOXO3a. Future studies are needed to clarify whether Sgk1 plays a role in regulating trpv6 expression.

The functional role of increased trpv6 expression was investigated using CdCl₂, a Trpv6 inhibitor (29). CdCl₂ treatment reduced NaR cell number in stc1a ^-/- fish to the sibling group levels ( Figure 3A ), indicating that Stc1a suppresses NaR cell proliferation by acting through Trpv6. If this were correct, then double deletion of Stc1a and Trpv6 should phenocopy each other. Indeed, compared to the siblings, the NaR cell number of trpv6 ^-/-; Tg(igfbp5a:GFP) fish was significantly higher. Double deletion of Stc1a and Trvp6 did not cause any further increase ( Figures 3B, C ), indicating that Stc1a and Trpv6 act via the same pathway. Akt is a downstream effector of IGF signaling and has been used as a proxy of IGF signaling in NaR cells due to the lack of antibodies to detected phospho-IGF1 receptors (31). To determine whether IGF signaling is involved, we measured phosphorylated Akt levels. Few Phospho-Akt positive cells were detected in wild-type and heterozygous siblings ( Figures 3D, E ). In comparison, a robust increase in Phospho-Akt positive NaR cells was detected in trpv6 ^-/- larvae ( Figures 3D, E ). The double stc1a ^-/-; trpv6 ^-/- mutant fish had a similar level of increase in Akt signaling as trpv6 ^-/- mutant fish ( Figures 3D, E ), suggesting that Stc1a and Trpv6 inhibit NaR cell proliferation via the same IGF signaling. It is worthy to point out the difference in the two approaches used to inhibit Trpv6 function/activity in this study. In Figure 3A , CdCl2 treatment was carried out in stc1a^-/- fish. These fish have a functional Trpv6 and at elevated levels. In this setting, CdCl2 treatment inhibited Trpv6-mediated calcium influx and resulted in reduced NaR cell proliferation, supporting the conclusion that Stc1a acts via Trpv6 to suppress NaR cell proliferation. In comparison, the experiment shown in Figures 3B, C used trpv6^-/- mutant larvae. In this genetic deletion model, there is no functional Trpv6 (29). Loss of Stc1a did not cause a further increase in NaR cell proliferation in the absence of a functional Trpv6. This result is in agreement with our conclusion.

It has been documented half a century ago that removal of CS resulted in increased body calcium contents and the appearance of kidney stones (4). This has been attributed to the loss of Stc1. This notion, however, has not been tested genetically due to the lack of a stable genetic mutant. We visited this issue using the stc1a ^-/- mutant fish. Compared to their wild-type and heterozygous siblings, abnormal calcium deposits were observed in the yolk sac region where NaR cells are located ( Figure 4A ). Highly calcified stone-like structures were also observed in the renal tube ( Figure 4A ). In a previous report, we have quantified the calcium levels in stc1a^-/- mutants and sibling embryos and found that stc1a^-/- fish had significantly elevated calcium levels (24). Taken together, these data suggest that a permanent loss of Stc1a results in calcium imbalance and the development of kidney stones, essentially recapitulating the classical experiment results reported by Pang in the 1970s (4) using molecular genetics in zebrafish. Are these abnormal calcium deposits and kidney stones observed in stc1a ^-/- larvae related to the increased trpv6 gene expression ( Figure 2 )? To address this question, we treated the fish with CdCl₂. CdCl₂ markedly reduced the calcified structures in the yolk sac region and in the renal tubes ( Figure 4A ). This was investigated further using double mutant fish. Alizarin red staining showed that the abnormal calcified structures were not observed in the stc1a ^-/-; trpv6 ^-/- double mutant fish. trpv6 ^-/- fish had markedly reduced staining as well ( Figure 4B ). These results suggest that Stc1a inhibits epithelial Ca²⁺ uptake by regulating Trpv6 expression and activity.

At 4-5 dpf, stc1a ^-/- mutants developed cardiac edema and this was followed by whole body swelling and premature death (24). In this study, we detected a significant reduction in heart rates ( Figure 5A ). These phenotypes are very different from the mouse model. Stc1^-/- null mice grew normally with no notable anatomical abnormalities (38). These differences among species may relate to their distinct physiology and different habitats. Zebrafish Stc1a is expressed and secreted from CS glands in a calcium concentration-regulated manner (10, 24, 39). Mice, however, do not have CS glands and Stc1 gene is expressed in many tissues and likely acts locally as a PAPP-A/PAPP-A2 inhibitor (2, 17). Mouse Stc1 does not appear to affect calcium homeostasis because Stc1 knockout mice had normal circulating calcium levels and normal Vitamin D3 response (38). Mice and other territorial animals take up Ca²⁺ from food and drinks. Zebrafish live in freshwater, a hypoosmotic aquatic environment (40). Zebrafish actively regulate their body osmolarity by maintaining ion water balance. They use ionocytes to uptake salts. At the same time, zebrafish remove the excess osmotic water by producing and excreting large volumes of diluted urine and reabsorbing ions in the kidney (39, 40). Although zebrafish nephrons begin to form, efficient glomerular filtration and ion re-absorption begin around 4-5 dpf (39, 41, 42). The cardiac edema and body swelling phenotypes observed in stc1a ^-/- mutant fish begin to manifest around 4-5 dpf. These led us to speculate that elevated epithelial Ca²⁺ uptake and impaired renal function may result in the accumulation of osmotic water, which lead to the progressive development of edema and swelling. If this were correct, then pharmacological or genetic blockade of Trpv6-mediated Ca²⁺ uptake should rescue the stc1a mutant fish. Indeed, treatment of stc1a^-/- fish with CdCl₂ alleviated the edema and body swelling phenotype ( Figure 5B ). While stc1a^-/- fish died between 6 to 10 dpf, there was no death in the CdCl₂ treated group until 10 dpf ( Figure 5C ). The role of Trvp6-mediated epithelial Ca²⁺ uptake was tested further by double knocking out stc1a and trpv6. As shown in Figures 5D, E , no cardiac edema or body swelling was observed in stc1a ^-/-; trpv6 ^-/- double mutant larvae. All stc1a mutant larvae lacked inflated swimming bladders ( Figure 5D ). This phenotype was rescued by CdCl₂ treatment ( Figure 5A ) but not by double deletion of stc1a ^-/- and trpv6 ^-/- ( Figure 5C ). The reason is not clear at this time. We have reported that the premature death can be rescued by reducing NaR cell number via pharmacological inhibition of the IGF1 receptor and Tor or by double deletion of igfbp5a or papp-aa in the stc1a-/- background (24). Since Stc1a and Trpv6 inhibit NaR cell number via the same IGF signaling, we tested the possible role of Trpv6 in zebrafish survival. While many stc1a ^-/- fish died between 7 to 10 dpf, no death was detected in stc1a ^-/-; trpv6 ^-/- fish, trpv6 ^-/- or siblings until 10 dpf ( Figures 5D, E ). These data suggest that the increased calcium uptake due to the combinatory effects of more NaR cells and great Trpv6 expression/NaR cell may cause ion water imbalance and premature death of stc1a ^-/- fish.

In summary, the results of this study have provided genetic and biochemical evidence that Stc1a regulates calcium homeostasis and organismal survival by playing dual roles in ionocytes ( Figure 6 ). Stc1a suppresses NaR cell proliferation via its reported role in inhibiting Papp-aa-mediated local Igfbp5a degradation (24, 26). Stc1a also inhibits Trpv6 expression and/or Trpv6-mediated calcium uptake ( Figure 6 ). These two functions are linked. While Trpv6-mediated calcium uptake inhibits IGF signaling, IGF signaling upregulates Trpv6 expression and stimulates NaR cell proliferation ( Figure 6 ). A loss of Stc1a results in a reactivation of IGF-PI3 kinase-Akt-Tor signaling in NaR cells, which stimulates NaR cell proliferation and increase NaR cell number and calcium uptake. In addition, loss of Stc1a also increases Trpv6 expression and Trpv6-mediated calcium uptake. These changes contribute to abnormal calcium deposits in the yolk sac region and in the kidney, the development of edema, body swelling, and premature death ( Figure 6 ). The current study also reveals a feedback loop from Trpv6 to Stc1a. While loss of Stc1a increases Trpv6 expression in NaR cells, loss of Trpv6 expression decreases Stc1a expression in CS. These findings provide new insights into our understanding of Stc1/STC1. At present, the biochemical pathways that lead to the formation of ectopic calcium deposits in the yolk sac region and in renal tubes found in the stc1a ^-/- mutant fish are not clear. In the adult stages, NaR cells are distributed mainly in the gills and kidney. Because stc1a^-/- mutant fish die prematurely, the function of Stc1a in adult physiology is not clear. A conditional knockout fish model will be needed to elucidate Stc1a’s actions in the adult gills, kidney, and intestine. In addition to stc1a, zebrafish have 3 other stc genes. Future studies will be needed to elucidate their functions and the relationship among these genes.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The animal study was approved by Institutional Committee on the Use and Care of Animals, University of Michigan. The study was conducted in accordance with the local legislation and institutional requirements.

SL: Writing – review & editing, Data curation, Formal Analysis, Investigation, Visualization. HL: Data curation, Formal Analysis, Investigation, Visualization, Writing – review & editing. ZW: Data curation, Formal Analysis, Investigation, Visualization, Writing – review & editing, Validation. CD: Writing – review & editing, Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – original draft.