Metabolic Diseases and Immunology Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa 50010
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ABSTRACT |
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The transcellular Ca2+ fluxes required for milk production must be rigorously regulated to maintain the low cytosolic Ca2+ concentrations critical to cell function. Ca2+-ATPases play a critical role in the maintenance of this cellular Ca2+ homeostasis. Using RT-PCR and sequencing, we identified six Ca2+ pumps in lactating mammary tissue. Three plasma membrane Ca2+-ATPases (PMCAs) were found (PMCA1b, PMCA2b, and PMCA4b). Two sarco (endo)plasmic reticulum Ca2+-ATPases (SERCAs) were identified (SERCA2 and SERCA3), and the rat homologue to the yeast Golgi Ca2+-ATPase RS-10 was also found. The pattern of mRNA expression of each of these pumps was examined in rat mammary tissue from the 7th day of pregnancy to the 21st day of lactation. Northern blots revealed increased mRNA expression for all Ca2+ pumps by the 14th day of lactation, and transcripts continued to increase through the 18th day of lactation. PMCA1b, PMCA4b, SERCA2, and SERCA3 showed the lowest levels of expression. RS-10 transcripts were more abundant than SERCA2, SERCA3, PMCA1b, and PMCA4b. RS-10 was the only pump to increase in expression before parturition. PMCA2b was the most abundant transcript found in lactating mammary tissue. At peak lactation, expression of PMCA2b approached that of actin. The high expression, high affinity for Ca2+, and high activity at low calmodulin concentrations exhibited by PMCA2b suggest that it is uniquely suited for maintenance of Ca2+ homeostasis in the lactating mammary gland. The pattern of expression and abundance of RS-10 suggest that it is a candidate for the Golgi Ca2+-ATPase shown to be important in maintaining the Golgi Ca2+ concentration required for casein synthesis and micelle formation.
breast; calcium pumps; lactation; plasma membrane calcium-adenosinetriphosphatase; sarco(endo)plasmic reticulum calcium-adenosinetriphosphatase
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INTRODUCTION |
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DURING LACTATION, the mammary gland transports large amounts of Ca2+ from the blood to milk via mammary alveolar cells. To prevent Ca2+ cytotoxicity, the large transcellular Ca2+ fluxes associated with lactation must be rigorously regulated and Ca2+ must be sequestered. This is a formidable task, as it has been estimated that the mammary gland in the cow stores enough Ca2+ for 12 h of milk production (40). The Ca2+ content of most extramammary tissues is <1 µmol/g tissue. In mouse mammary tissue, Ca2+ increases from 2 µmol/g tissue during pregnancy to 12 µmol/g tissue at the initiation of lactation (30, 32), and values of 30-40 µmol Ca2+/g mammary tissue (6) have been reported in lactating cows. In the cow, this rapid influx and storage of Ca2+ in the mammary gland can cause a near fatal hypocalcemia at parturition (23, 35). This large intracellular Ca2+ pool in the mammary gland is thought to be concentrated in and around the Golgi apparatus (9). Millimolar Ca2+ in the endoplasmic reticulum (ER) and Golgi are essential for normal protein synthesis, processing, and secretion (14, 41). Rat and cow milk are 60 and 30 mM Ca2+, respectively. Two-thirds of this Ca2+ arrives in milk secreted as a part of the casein micelle (29, 32). Therefore, a mechanism must be in place to support this high Ca2+ flux into the ER and Golgi. In addition, milk contains 1-4 mM free Ca2+, which must be moved up a concentration gradient from the cell to milk. The remaining Ca2+ arrives associated with citrate (29, 32).
The task of maintaining mammary cell Ca2+ homeostasis while moving large amounts of Ca2+ through the secretory cell has received limited investigation. Biochemical evidence exists for a P-type Ca2+-ATPase in the Golgi of mammary tissue with characteristics slightly different from either the plasma membrane Ca2+-ATPase (PMCA) or the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) (6, 7, 14, 31, 43). This mammary Golgi Ca2+-ATPase could be a homologue of the yeast Golgi Ca2+-ATPase PMR1 (15, 37). A rat homologue (clone RS-10) of the yeast Ca2+-ATPase has been cloned, but its function is unproven and its cellular location remains to be determined (19-21).
The Ca2+-ATPase families PMCA and SERCA are similar but distinct (27). The PMCAs are located in the plasma membrane, form an efficient system for pumping Ca2+ from the cell, and are responsible for maintenance of intracellular Ca2+ levels. Mammalian PMCAs are encoded by a multigene family consisting of four members termed PMCA1, PMCA2, PMCA3, and PMCA4 (10-12). Additional diversity is generated by alternative RNA splicing (2, 11, 18, 22, 25, 28, 39). Alternative splicing of the COOH-terminal tail has been shown to modify the regulatory properties of PMCA isoforms, particularly with respect to calmodulin stimulation (16, 17). Many PMCA isoforms and splice variants are expressed in a tissue- and cell-specific manner. PMCA1 and PMCA4 are ubiquitously expressed and are thought to be housekeeping forms of the pumps. PMCA2 and PMCA3 have more restricted patterns of expression and are found primarily in nervous tissue (11). In the kidney and intestinal epithelia, PMCAs are involved in macro transcellular Ca2+ flux, with the pump localized to the basolateral membrane (8, 44). These observations provide a precedent for PMCA involvement in the macro transcellular Ca2+ fluxes needed for milk production. The SERCA family of Ca2+-ATPases are located in the ER, and they work with the PMCAs to remove Ca2+ from the cytoplasm and set resting cytoplasmic Ca2+ concentrations. The ER is a major intracellular storage site for Ca2+ that can be released by various agonists; ER Ca2+ is then replenished by the action of the SERCAs. The SERCAs are encoded by a multigene family consisting of three members termed SERCA1, SERCA2, and SERCA3 (26). As with PMCAs, additional diversity is generated by alternative splicing. SERCA1a and SERCA1b are exclusively expressed in fast-twitch skeletal muscle and therefore are not a concern for mammary function. SERCA2a is the cardiac and slow-twitch muscle isoform, and SERCA2b is expressed in both smooth muscle and nonmuscle tissues. SERCA3 is expressed in a restricted set of nonmuscle tissues, including epithelial and lymphocytic cells (26).
In this study, we used RT-PCR and sequencing of the PCR products to determine which Ca2+-ATPases are expressed in the lactating mammary gland. Subsequently, we used Northern blotting to quantitate the expression of these Ca2+-ATPase mRNAs in mammary tissue during pregnancy and lactation. The timing and amount of transcript expressed suggested that the putative rat Golgi Ca2+-ATPases (RS-10) could represent some of the Ca2+-ATPase associated with the mammary Golgi system. Additionally, the timing and amount of transcript expressed for PMCA2b suggested that this pump is the major Ca2+-ATPase involved in macro Ca2+ homeostasis in the lactating rat mammary gland.
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METHODS |
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Animals.
All animal procedures were approved by the National Animal Disease
Center Animal Care and Use Committee. Sprague-Dawley rats confirmed to
be pregnant were purchased from Harlan Sprague Dawley (Madison, WI).
Rats were housed individually in hanging basket cages on sawdust
bedding. Beginning 7 days after the breeding date, rats were
killed 14, 7, and 1 day prepartum and 7, 14, 18, and 21 days postpartum. Data (see Figs. 3-7) are presented in days, with
parturition as the reference point: negative days indicate days before
parturition and positive days indicate days of lactation (i.e., after
parturition). Rats were anesthetized with a 50:50 mixture
of CO2 and
O2 and then decapitated. Blood was
collected to prepare serum for the assay of
Ca2+ by atomic absorption
spectroscopy and 1,25-dihydroxyvitamin
D3 [1,25(OH)2D3]
(36). Rats were confirmed to be pregnant by inspection of the uterus.
Mammary tissue was removed, flash frozen in liquid N2, and stored at 70°C
until RNA was prepared.
RNA preparation. Total RNA and DNA were isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction (13) using TRIzol reagent (GIBCO-BRL, Gaithersburg, MD). Poly(A)+ RNA was prepared using PolyATract mRNA Isolation System IV (Promega, Madison, WI).
RT-PCR and sequencing.
Primers (Table 1) were selected for isoform
or splice form specificity, and many of the primers used here have been
previously described (1, 42, 45). PCR was carried out in a GeneAmp 9600 PCR system (Perkin Elmer, Foster City, CA). The RT-PCR reaction master
mix (99 µl) consisted of 10 µl of 10× reaction buffer
(Boehringer-Mannheim, Indianapolis, IN), 77.75 µl of diethyl
pyrocarbonate-treated water, 8 µl of dNTPs (from 10 mM stock;
Amresco, Solon, OH), 1 µl of each of the specific primers (100 pmol/µl; IDT, Coralville, IA), 0.25 µl (50 units) Moloney murine
leukemia virus (GIBCO-BRL), 0.5 µl (20 units) RNAsin (Promega), and
0.5 µl (2.5 units) Taq polymerase
(GIBCO-BRL). To this master mix was added 1 µl of template (1 µg
RNA).
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Northern blot analysis.
Poly(A)+ RNA (2-3 µg) was
fractionated in formaldehyde-agarose gels, and blotting and
hybridization were performed as previously described (24). PMCA1 and
RS-10 cDNAs were a kind gift from Gary Shull (University of Cincinnati,
Cincinnati, OH). All other cDNA probes were prepared by RT-PCR using
the primers specified in Table 1. After hybridization, the blots were
imaged and quantitated on an Instantimager (Packard Instruments,
Downers Grove, IL). All Northern blot data were normalized to -actin
expression. All transcript sizes were used in the quantitation of each pump.
Statistical analysis. Differences in plasma Ca2+, 1,25(OH)2D3, or pump expression as affected by day were analyzed by least squares ANOVA. When day effects were significant, specific comparisons were made using Tukey's test. All analyses were performed using Statview (SAS Institute, Cary, NC).
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RESULTS |
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Identification of
Ca2+-ATPase
isoforms and splice forms in lactating mammary tissue.
Total RNA was isolated from rat lactating mammary tissue and subjected
to RT-PCR using the PMCA isoform-specific primers described in Table 1.
Note that this table shows the expected PCR product sizes for each
primer pair. PMCA1, PMCA2, and PMCA4 isoforms were found to be present
in lactating mammary tissue, and their identities were confirmed by the
product sizes (835, 427, and 563 bp, respectively; Fig.
1,
left) and by
sequencing of each PCR product (data not shown). We also determined the
splice form for each PMCA found. The b splice form was found for all
three PMCAs (Fig. 1, right) on the
basis of PCR product sizes (431, 560, and 213 bp, respectively) and
sequencing (data not shown). On the basis of PCR product size (388 bp),
we initially thought we had found PMCA4a; however, we could not confirm
the presence of PMCA4a by sequencing.
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Effect of mammary gland development and lactation on rat
Ca2+ metabolism.
Blood samples from female rats at various stages of pregnancy and
lactation were assayed for plasma
Ca2+ and the calcitropic hormone
1,25(OH)2D3
as indicators of loss of body Ca2+
to milk production. One day before parturition, a significant (P < 0.05) hypocalcemia
occurred in conjunction with the rat's preparation to give birth and
initiate lactation (Fig. 3,
top). Plasma
1,25(OH)2D3
increased in response to the hypocalcemia to stimulate intestinal
Ca2+ transport in support of
lactation (Fig. 3, bottom) and had
significantly (P < 0.01) increased 1 day before parturition.
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General transcriptional changes in rat mammary gland as it prepares
for milk protein synthesis.
Mammary tissue DNA per gram tissue remains relatively constant through
pregnancy and lactation (data not shown). However, micrograms RNA per
gram tissue (data not shown) and micrograms RNA per microgram DNA (Fig.
4, top)
rise rapidly as lactation approaches. These dramatic changes must be
taken into account when presenting
Ca2+-ATPase mRNA data in this
rapidly changing tissue. Because of this large increase in RNA
synthesis by the lactating gland, specific mRNAs, such as actin, can
appear to be declining when they remain unchanged or are actually
increasing slightly (Fig. 4,
bottom). Therefore, all subsequent
measures of specific Ca2+-ATPase
mRNA expression during this period take this dilution effect into
account. For simplicity, all
Ca2+-ATPase mRNA data presented
were corrected for actin expression. Correction of mRNA data on a per
gram tissue or per milligram DNA basis yielded data almost identical to
those adjusted for actin as presented here.
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Ca2+-ATPase
expression in rat mammary tissue as affected by pregnancy and
lactation.
The pattern of mRNA expression of each
Ca2+-ATPase was examined in rat
mammary tissue from the 7th day of pregnancy to the 21st day of
lactation. Northern blots revealed increased mRNA expression for all
Ca2+ pumps by the 14th day of
lactation, and transcripts continued to increase through the 18th day
of lactation (Figs.
5-7).
PMCA1b, PMCA4b, SERCA2, and SERCA3 showed the lowest levels of
expression during both pregnancy and lactation and only became
significantly elevated (P < 0.05) on
the 14th day of lactation (Figs. 5 and 6). PMCA1b, SERCA2, and SERCA3
transcripts increased only modestly to two and four times their
prepartum levels. PMCA4b expression rose 10 times during the same time
frame, but none of these
Ca2+-ATPases approached the levels
of expression observed for PMCA2b and RS-10.
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DISCUSSION |
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The many Ca2+-ATPases present in cells are essential for normal cell physiology. They control intracellular Ca2+ balance required for signal transduction by pumping Ca2+ out of the cell or into cell organelles. In the intestine and the kidney, PMCAs also play primary roles in transcellular transport of macro amounts of Ca2+ (5, 8, 44). The role, if any, of these Ca2+-ATPases in macro Ca2+ movements within a specialized Ca2+-secreting cell, such as lactating mammary cells, is unknown. As a first step to address the role of Ca2+-ATPases in Ca2+ movement in the lactating mammary gland, we used RT-PCR sequencing to determine which Ca2+-ATPases were present in the lactating mammary gland.
We found five Ca2+-ATPases and one putative Ca2+-ATPase (RS-10) to be expressed in mammary tissue. The ubiquitously expressed housekeeping isoforms (11) PMCA1b and PMCA4b were found in mammary tissue and are likely expressed in all of the various cell types in mammary tissue. PMCA4a appeared at first to be present, but this could not be confirmed by sequencing. No evidence of the specialized PMCA3 isoform was found. However, PMCA2b, which has been shown to be expressed only in a limited number of tissues (11), was found, primarily after the start of lactation, thus indicating that the primary site of expression for PMCA2b may be the secretory epithelial cell. SERCA2, which is thought to be essential (26) for cell function, was present, as was SERCA3, which is only expressed in a limited number of tissues (26). SERCA2 would be found in all cells of the mammary gland, since it is essential for cell function (26). Finally, RS-10, the ubiquitously expressed mammalian homologue of the yeast secretory pathway Ca2+ pump, was found (20). Its increased expression as lactation starts indicates a specialized role for RS-10 in the secretory cell.
Understanding the factors regulating Ca2+ transport, storage, and secretion from the mammary gland has several implications for both animal health and mammary physiology. As is seen in Fig. 1, a transient hypocalcemia occurs 1 day before parturition, as the mammary gland fills with Ca2+ for milk production. In most mammals, this hypocalcemia due to lactation is of little concern. In the dairy cow, this process can lead to life-threatening hypocalcemia with metabolic complications (23, 35). The rapid rise in plasma 1,25(OH)2D3 (Fig. 3) is a normal response to the Ca2+ needs of an animal, and 1,25(OH)2D3 has been shown to increase the expression of PMCA1 in the intestine (5, 44). The mammary gland has receptors for 1,25(OH)2D3 (34); however, the observed rise in plasma 1,25(OH)2D3 is not likely to affect Ca2+-ATPase expression in the mammary gland, as it has been shown that 1,25(OH)2D3 plays little or no role in mammary Ca2+ homeostasis (3, 23).
In mouse mammary tissue, Ca2+ increases from 2 µmol/g tissue during pregnancy to 12 µmol/g tissue at the initiation of lactation (30, 32), whereas values of 30-40 µmol Ca2+/g mammary tissue (6) have been reported in the mammary tissue of the lactating bovine. It is thus not surprising that expression of all six Ca2+-ATPases increased during the lactation period (Figs. 5-7). Clearly, the control of intracellular Ca2+ essential to normal cell function requires the concerted effort of all these Ca2+-ATPases.
Of particular interest were both the presence and level of PMCA2b expression in the lactating mammary gland (Fig. 7). Before acquisition of the data presented here, PMCA2 had only been observed in significant amounts in brain and nervous tissue. By PCR, PMCA2 has been detected in heart, skeletal tissue, kidney, testes, and pancreas (2, 25, 42). However, when these tissues were examined for quantitative expression of PMCA2 mRNA or protein, little or no PMCA2 was detected (38, 39). The lactating mammary gland is the first nonnervous tissue to express significant amounts of PMCA2 mRNA. Some of the unique characteristics of PMCA2 make this Ca2+-ATPase uniquely suited for maintenance of Ca2+ homeostasis in the environment of high Ca2+ flux of the mammary gland. Elwess et al. (16) showed that PMCA2b has the highest affinity for both Ca2+ and calmodulin of all the PMCAs tested to date. PMCA2b also showed higher basal activity in the absence of calmodulin. From these data, the authors concluded that PMCA2b would be extremely effective at lowering intracellular Ca2+ and thus be very important in the cells where it is expressed. Its extremely high expression in the lactating mammary gland suggests that PMCA2b plays a significant role in the Ca2+ homeostasis of this tissue.
On a more speculative note, both the timing and magnitude of RS-10 expression in lactating mammary tissue are interesting. Of all the Ca2+-ATPases studied here, this putative Ca2+-ATPase (RS-10) (20) is the only transcript that increased significantly before parturition (Fig. 7). This is significant because that is the time when the animal becomes hypocalemic due to rapid influx of Ca2+ to mammary stores (Golgi). Clear biochemical evidence exists for a Golgi Ca2+-ATPase important to mammary function in general and to casein micelle synthesis in particular (6, 7, 14, 30-32, 43). The yeast equivalent of RS-10, PMR1, is clearly a Golgi Ca2+-ATPase (4, 15, 33, 37). Unfortunately, Shull's group (20) has not been able to demonstrate Ca2+-ATPase activity for RS-10 or localize it to the Golgi. An antibody for RS-10 will be needed for such studies. An argument against RS-10 as the mammalian Golgi Ca2+-ATPase has been presented (41). These authors suggest that there is no unique mammalian Golgi Ca2+-ATPase but rather that SERCAs in the ER and PMCAs in transit to the plasma membrane maintain the Ca2+ concentrations found in the Golgi. Further studies on the location and function of RS-10 are warranted, as we have found that RS-10 is expressed in large amounts 7 days before lactation in the cow and that its expression is highly correlated with the degree of hypocalcemia the cows develop (S. Prapong, R. Horst, and T. Reinhardt, unpublished observations).
In conclusion, the high expression, high affinity for Ca2+, and high activity at low calmodulin concentrations exhibited by PMCA2b suggest that PMCA2b is uniquely suited for maintenance of Ca2+ homeostasis in the lactating mammary gland. RS-10, because of its pattern of expression and abundance, is a candidate for the Golgi Ca2+-ATPase shown to be important in maintaining the Golgi Ca2+ concentration required for casein synthesis and micelle formation in the lactating mammary gland.
Further studies to understand the role of each pump in mammary Ca2+ homeostasis are needed. Are the PMCAs on the apical membrane of the secretory cell or on the basolateral membrane, as shown in intestine and kidney? Can RS-10 be localized to the Golgi and shown to be a true Ca2+-ATPase, or is the large amount of PMCA2b being made contributing to Golgi Ca2+ as it transits to the plasma membrane? Most importantly, are the apparently high levels of mRNA expression for RS-10 and PMCA2 ultimately reflected in high levels of protein expression? New general and specific antibodies will help to address some of these issues.
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ACKNOWLEDGEMENTS |
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We thank Becky Zaworski, Becky Baccam, and Derrel Hoy for excellent technical assistance and Annette Bates for expert preparation of the manuscript.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. A. Reinhardt, Metabolic Diseases and Immunology Research Unit, National Animal Disease Center, ARS-USDA, Ames, IA 50010 (E-mail: treinhar{at}nadc.ars.usda.gov).
Received 31 August 1998; accepted in final form 22 December 1998.
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