SPCA1 and SPCA2 are both Golgi secretory pathway Ca2+/Mn2+-ATPases. However, SPCA2 has a more restricted tissue expression than the housekeeping form SPCA115. Both SPCA1 & 2 are highly expressed in mammary tissue and the expression of both increases massively with milk production6,16,17. Functionally we know little about SPCA1 in lactation in part because SPCA1 knockout mice are not viable18 and SPCA1 heterozygotes did not have a significant change in milk or mammary phenotype Reinhardt et al. unpublished). However, SPCA2 was found to increase the influx of calcium into breast tumor cells by SICE10 and subsequent in vitro experiments with a cell model of lactation demonstrated that SPCA2 and Oria1 together mediate the basolateral calcium influx into mammary secretory cells to support the large calcium requirements of lactation via SICE6. In contrast, using Orai1 knockout mice, it was determined that Orai1 and the store-operated mechanism (SOCE) of calcium influx to secretory cells is responsible for up to 50% of the calcium transported into milk9. The current availability of a SPCA2 knockout mouse model has allowed us to examine the role of SPCA2 in lactation.
So, the general working hypothesis was that loss of SPCA2 (Fig. 1B) would at a minimum lead to reduced calcium in milk8 due to reduced calcium entry into the secretory cells thus, providing less available calcium for secretion with milk. The loss of SPCA2 to partner with Orai1 resulted in SICE loss and a 40 % reduction in milk calcium (Fig. 6A). Therefore, SOCE is responsible for 50% of the calcium transported into milk9 and SICE is responsible for 40%. That accounts for most of the calcium influx into the secretory cells via these two pathways. However, these in vivo gene deletion lactation models are not unambiguous enough to allow these conclusions. The Orai1 gene deletion paper did not consider the possibility of Orai1 partnering with SPCA2 to support SICE6,9,12. Only SOCE was considered as a mechanism for calcium influx into secretory cells and they noted that neither Orai2, Orai3, nor Stim1or Stim2 showed compensatory upregulation due to loss of Orai19. Similarly, there was no or little compensatory upregulation most components of CALTRANS due to loss of SPCA2 with the notable exception of SPCA1 (Figs. 1, 2 and 3)
In non-gene deletion mouse lactation experiments, it has been shown that Orai1 expression rises as milk production increases, whereas Stim1 expression declines precipitously19. This might suggest that a classical partnering of Orai1 and Stim1 to support mM SOCE in lactation is not viable. However, Stim2 is upregulated with lactation, thus providing a possible partner to support SOCE. In contrast, our data support the SICE pathway. At this point, we can only conclude without further information that both SICE and SOCE contribute to the massive influx of calcium required to support normal milk production. Other calcium ion channels that regulate this enormous calcium influx may be necessary to support normal milk calcium, but this information awaits further research.
The primary lactation defect associated with loss of SPCA2 was reduced milk production as estimated by pup weights (Fig. 4D). Reduced milk production seems to be a common lactation defect when some CALTRANS genes are knocked out4,8,9. Histologically, the loss of SPCA2 resulted in mammary tissue adipocytes (Fig. 5E), a significant increase in % adipose tissue (Fig. 4F) and a measured reduction in tissue membrane content (Fig. 4E). All these likely contribute to the loss of milk production. We also observed that loss of SPCA2 lead to mammary alveoli with missing epithelial cells that allowed milk to leak into the interstitial spaces (Fig. 5E). This observation suggested loss of normal epithelial tissue structure and integrity, which is in part a function of E-Cadherin20. Recent work from Rao’s lab21 demonstrated that SPCA2 expression positively correlates with E-cadherin expression in breast cancer cells. Furthermore, in these breast cancer cells, loss of SPCA2 with the concomitant reduction in E-cadherin promoted an epithelial to mesenchymal cell transition, which might explain our apparent loss of epithelial tissue structure in mammary alveoli (Fig. 5E). However, in normal lactating mammary tissue, this relationship between SPCA2 and E-cadherin seems to be missing (Fig. 3D). The downregulation of the ZO-1 tight junction protein was the only observed protein change in SPCA2 knockouts that might contribute to the loss of epithelial tissue structure in mammary alveoli (Fig. 5E). Further work will need to be done to address this effect of SPCA2 loss.
Milk components such as protein, fat and lactose were unaffected in the SPCA2 knockout mice. This contrasted with the significant reduction of lactose and increase in milk protein in PMCA2 and TMEM165 KO4,8. The lactose reduction in PMCA KO mice was transient with no explanation. However, the reduction in lactose synthesis seen in TMEM165 was partly attributed to a marked decrease in manganese transport to milk in TMEM165 KO’s that suggested reduced Golgi Manganese. Since manganese is a cofactor for lactose synthetase, it was concluded that, in part, the lack of manganese might have contributed to the reduced lactose seen in milk from TMEM165 KO8. Since a similar reduction in milk manganese was observed with the SPCA2 knockout (Fig. 6B) and milk lactose was not affected, we might assume that the TMEM165 effect on milk lactose was due to other factors such as TMEM165 proposed role in the regulation of Golgi pH8.
In this SPCA2 KO experiment, there was some compensatory regulation of proteins that make up the CALTRANS module of calcium pumps, channels, transporters, sensors, binding proteins, and buffers12,13. Both SPCA1 and Stim 1 were moderately upregulated. Similar compensatory regulation was found following knocking out the PMCA2 gene, which resulted in compensatory upregulation of SPCA1 and SERCA2 in lactating mammary tissue4. A cell-based system overexpression of SPCA1 in COS-7 cells resulted in compensatory downregulation of pan-PMCA, calreticulin and CALNUC22. Despite compensatory upregulation of SPCA1 and Stim1 they were unable to compensate for the loss of SPCA2. SPCA1 as a Ca2+/Mn2+ transporter clearly could not make up for the milk/cell Mn2+ deficit induced by loss of SPCA2.
To screen for members of CALTRANS more efficiently, we used a label-free proteomics approach to quantitate changes in the proteome between WT and KO mammary membranes, mammary whole cell lysates and milk whey. To our surprise, we found almost no compensatory regulated CALTRANS members with the loss of SPCA2 from lactating mammary glands (Supplementary Tables 1, 2 & 3 imputed tabs). Most of the differentially regulated proteins due to loss of SPCA2 could not, with our current knowledge, be directly related to the loss of SPCA2. The lone exception may be significant downregulation of potential CALTRANS member Inositol 1,4,5-trisphosphate receptor type 1 (Supplementary Table 3 imputed tab), which mediates the mediates calcium release from the endoplasmic reticulum. A reason for its downregulation would be speculation.
Osteopontin was found to be significantly upregulated following the loss of SPCA2 (Supplementary Tables 1 & 2 imputed tabs) in two of the proteome datasets. Osteopontin is an adhesive extracellular matrix protein whose loss results in abnormal mammary morphogenesis and defective lactation23. In contrast, in this study, Osteopontin is overexpressed following the loss of SPCA2. Adipose tissue is a known source of Osteopontin24 and since the SPCA2 knockout tissue has significantly more adipose tissue (Fig. 4F&5E), this could be the reason we see more Osteopontin in SPCA2 KO mice. In breast cancer cells, Osteopontin increases both cell migration, cell invasiveness and metastasis25,26. Thus, the higher Osteopontin expression seen in SPCA2 KO could contribute to changes in mammary tissue described in Fig. 5E.
In conclusion, the significant decline in milk calcium following the loss of SPCA2 supports a role for SPCA2 in SICE. The loss of SPCA2 to partner with Orai1 resulted in a 40 % reduction in milk calcium. This significant decline in milk calcium following the loss of SPCA2 supports a role for SPCA2 in SICE in lactation with in vivo data. Additional studies will be required to clarify this relationship between SPCA2, ORAI1, and STIM1 on mammary calcium influx in vivo and the relative role of SICE and SOCE in mammary calcium influx to support calcium needs for lactation.