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Am J Physiol Regul Integr Comp Physiol 281: R1169-R1176, 2001;
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Vol. 281, Issue 4, R1169-R1176, October 2001

Na+-K+-ATPase gene expression in the avian eggshell gland: distinct regulation in different cell types

Irena Lavelin, Noam Meiri, Olga Genina, Rosaly Alexiev, and Mark Pines

Institute of Animal Sciences, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The avian eggshell gland (ESG) is a tissue specialized in transporting the Ca2+ required for eggshell formation and represents a unique biological system in which the calcification process takes place in a circadian fashion. With the use of RNA fingerprinting, a set of genes differentially induced at the time of calcification was detected, one of which was identified as the alpha 1-subunit of Na+-K+-ATPase. The gene was expressed in a circadian manner in both cell types populating the ESG, but in different temporal patterns, suggesting distinct mechanisms of regulation. Ca2+ flux and mechanical strain were found to regulate gene expression in the inner glandular epithelium and the pseudostratified epithelium facing the lumen, respectively. Mechanical strain also affected gene expression in cell layers facing the lumen in other parts of the oviduct. Only the alpha 1-isoform, not the alpha 2- or alpha 3-isoform, of Na+-K+-ATPase was expressed in the ESG. In summary, we demonstrate that the alpha 1-subunit Na+-K+-ATPase gene is expressed in different epithelial cell types in the ESG and is regulated by various mechanisms, which may reflect the disparity in the physiological roles of the cells in the process of eggshell formation.

mechanical strain; calcium flux; acetazolamide; isoforms


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AVIAN EGGSHELL FORMATION REPRESENTS a unique biological system in which the calcification process takes place in a circadian fashion. The eggshell is formed during passage of the egg through the oviduct, where the different layers of the eggshell are assembled sequentially. After fertilization of the ovum, the egg spends 2-3 h in the magnum, where the proteins for embryo consumption are secreted, and 1-2 h in the isthmus, where the two egg membranes are built. The egg then enters the eggshell gland (ESG) for an additional 18-20 h, where Ca2+ deposition occurs. During Ca2+ transport from the plasma to the lumen of the ESG for shell formation, ~10% of the body's total Ca2+ is secreted within 18-20 h. This makes eggshell formation one of the most rapid biomineralization processes known (14, 54). The deposition of calcium carbonate is accompanied by large changes in the flux of K+, Mg2+, and glucose into and Na+ and Cl- out of the ESG fluid, while they recirculate within the ESG mucosa via an active transport system. The Ca2+ flux is probably an active process (18, 45). The presence of Ca2+-ATPase in the ESG and its localization in the tubular gland cells (59) suggest the involvement of a Ca2+ pump in Ca2+ transport. The capacity of the ESG tissue to transport Ca2+ is highly correlated with the concentration of the vitamin D-dependent Ca2+-binding protein calbindin (13, 42). The calbindin gene is expressed in the ESG epithelium in circadian fashion, correlated with the egg's daily cycle (33, 59).

In the ESG, the genes encoding proteins that play a role in the control of eggshell calcification have been found to be regulated by hormonal (12, 29, 43, 55) and nonhormonal stimuli (3, 33, 55). Ca2+ flux is considered to be one of the major nonhormonal stimuli affecting ESG gene expression. For example, changes in Ca2+ flux caused by a carbonic anhydrase inhibitor (3, 33) or manipulation of shell formation without alteration of gonadal hormones (43, 44) caused changes in calbindin gene expression. Recently, we proposed that the mechanical strain imposed by the egg entering the ESG might also affect gene expression of eggshell proteins such as osteopontin (OPN) (33, 48). Strain regulation of OPN gene expression is part of the normal physiology of the laying hen and is applied in a circadian fashion during the daily egg cycle.

In this study, we used an RNA fingerprinting technique to search for more genes involved in eggshell calcification and regulated by mechanical strain. The unique circadian fashion of eggshell calcification allowed us to compare ESG gene expression at different physiological stages: 1) no calcification and no mechanical strain and 2) peak calcification and maximal mechanical strain. Moreover, perturbation of the system is possible by changing the rate of Ca2+ flux or the magnitude of the mechanical strain. One of the genes identified by RNA fingerprinting was the alpha 1-subunit of Na+-K+-ATPase (alpha 1NKA).

Na+-K+-ATPase is an enzyme responsible for maintaining the low internal Na+ and high internal K+ concentrations typical of most vertebrate cells. The ion gradients created by the enzyme are important in preserving the volume, pH, and electrical resting potential of cells (27, 28). The enzyme consists of two subunits necessary for its normal function. The alpha -subunit is a multispanning membrane protein that contains the binding sites for ATP, the cations, and the specific inhibitor ouabain, and the beta -subunit is a type II glycosylated polypeptide that crosses the membrane only once (34, 37). Because numerous cellular transport systems are coupled to the movement of Na+, Na+-K+-ATPase activity has adapted to various physiological requirements in different cell types. One way to achieve such specificity is the expression of various isoforms of the enzyme that differ in their affinities for cations and ATP, essential for cellular activities in specific physiological settings (4). The Na+ gradient established by the enzyme activity provides the energy to fuel the secondary transport systems that mediate the translocation of various ions, one of which is Ca2+. Ca2+ ions also regulate the mechanical strain-dependent activation of the alpha -subunit of the enzyme.

This study describes the regulation of alpha 1NKA gene expression in the ESG of hens by Ca2+ and mechanical strain during the daily cycle of eggshell formation.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals, inhibition of calcification, and mechanical strain induction. Female Loman chickens, 4-8 mo of age, were used for all experiments. Samples of magnum, isthmus, ESG, and colon were collected at various intervals of the egg cycle. Samples were frozen in liquid nitrogen for RNA extraction or fixed overnight in 4% paraformaldehyde at 4°C for in situ hybridization. Acetazolamide (Sigma, St. Louis, MO) was orally administered (35 mg/kg body wt) 3-4 h after oviposition. This carbonic anhydrase inhibitor interferes with normal CO2 metabolism and with the transfer of carbonate needed as a counterion in the formation of the calcite eggshell. Administration of this drug results in significant inhibition of eggshell formation and causes a reduction in shell thickness (3, 36). Mechanical strain was induced by insertion of an endotracheal tube with a volume of 60 cm3, resembling the size and shape of an egg, into the ESG 3 h after oviposition, as previously described (33).

RNA fingerprinting. Total RNA was extracted with TriReagent (MRC, Cincinnati, OH) and fingerprinted with a Delta RNA fingerprinting kit (Clontech, Palo Alto, CA) using dATP (Radiochemical Center, Amersham). First-strand cDNA was synthesized using 2 µg of total RNA as a template, oligo(dT) as a primer, and Moloney's murine leukemia virus reverse transcriptase (GIBCO BRL Life Technologies). Two dilutions of each cDNA template (corresponding to 5 and 20 ng of reverse-transcribed RNA) were used for the PCR. In addition, each PCR contained 50 µM dNTPs, 1 µM primers, 50 nM [alpha -33P]dATP (1,000-3,000 Ci/mmol), and 1 µl of Advantage KlenTaq polymerase (PE Applied Biosystems, Foster City, CA). PCR primers were a pairwise combination of arbitrary "P" and oligo(dT) "T" primers (see reference manual for oligonucleotide sequences; Clontech, Palo Alto, CA). The thermal cycles were as follows: one cycle of 94°C, 40°C, and 68°C for 5 min each; two cycles of 94°C for 2 min, 40°C for 5 min, and 68°C for 5 min; and 22 cycles of 94°C for 1 min, 60°C for 1 min, and 68°C for 2 min. The PCR products were electrophoresed on a 5% acrylamide-8 M urea gel and run in 0.1 M Tris · borate-2 mM EDTA buffer. In a typical RNA fingerprint, ~80-100 bands were evident at each amplification. Differentially expressed cDNAs were eluted from the gel, reamplified, subcloned into a pGEM-T Easy cloning vector (Promega, Madison, WI), and sequenced. Nucleotide sequences were subjected to FASTA search for sequence homologies.

RT-PCR. To identify chicken alpha 1NKA and 18S mRNAs by RT-PCR, forward (F) and reverse (R) primers were used as follows: 5'-CAAGTCAGCCCACTGCACTA-3' (F) and 5'-GCTCAGATGTGTCCAAGCAA-3' (R) for alpha 1NKA and 5'-CCGAGGACCTCACTAAACCA-3' (F) and 5'-AGTTGGTGGAGCGATTTGTC-3' (R) for 18S. Expected amplification products were 615 bp for alpha 1NKA and 398 bp for 18S. To differentiate among three isoforms of the alpha -subunit of Na+-K+-ATPase, a set of four primers was used: 5'-GCCTCTTCATGATGTCACTCTC-3' (R) for all three isoforms and 5'-AGTTCCCTGAAGGCTTCCAG-3' (F) for alpha 1, 5'-GAGGTCAACTTCCCCACCAG-3' (F) for alpha 2, and 5'-ACTTTGCGACGGACAATCTG-3' (F) for alpha 3. Expected amplification products were 791, 781, and 765 bp, respectively.

RNA from ESG was reverse-transcribed to single-strand cDNA using oligo(dT) primers and Moloney's murine leukemia virus reverse transcriptase. A PCR master mix was added to the cDNA reaction to yield the following final concentrations: 1 µM specific primers, 200 µM dNTPs, 2.5 U of Taq DNA polymerase, and Taq buffer containing 1.5 mM MgCl2. PCR was performed for 30 cycles (94°C for 20 s, 60°C for 30 s, and 68°C for 1 min). Amplification products arising from RT-PCR were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining.

Preparation of riboprobe, in situ hybridization, and Northern blot. A fragment of cDNA corresponding to bases 2245-2859 of the chicken alpha 1NKA mRNA (GenBank accession no. J03330) was synthesized by RT-PCR from the chicken ESG. This fragment shared no homology with any other known proteins in the ESG and was subcloned into a pGEM-T Easy cloning vector to produce a template for riboprobe synthesis. The sense and antisense riboprobes were synthesized by in vitro transcription with T7 or SP6 RNA polymerase, respectively, in the presence of linearized plasmid DNA and DIG RNA labeling mix. Serial 5-µm sections were hybridized with digoxigenin-labeled avian alpha 1NKA probe as described previously (33). All materials for preparing the digoxigenin-labeled probes for in situ hybridization were obtained from Boehringer Mannheim (Ottweiler, Germany). No hybridization was observed with the sense riboprobes used as negative control. For the Northern blot analysis, ESG RNA (15 µg) was denatured, electrophoresed on a 1% agarose-formaldehyde gel, and transferred to 0.2-µm Nytran membranes. The RNA blots were hybridized with 32P-labeled cDNA probe for alpha 1NKA and S18 at 42°C in hybridization buffer containing 50% formamide.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of the alpha 1NKA gene by differential display. To identify genes involved in eggshell formation, we used biopsies of ESG from two different physiological states: 1) no egg residing in the ESG, no calcification occurring, and 2) an egg residing in the ESG, at the peak of eggshell calcification. Each group consisted of three different birds. RNA was reverse-transcribed and then fingerprinted using different pairs of arbitrary selected primers. Clones that appeared upregulated at the time of eggshell calcification were collected. We isolated and sequenced 14 candidate cDNAs, one of which, a 510-bp cDNA fragment, was found to be identical to alpha 1NKA (GenBank accession no. J03230).

Temporal and spatial expression of the alpha 1NKA gene during eggshell formation. Northern blot analysis of alpha 1NKA mRNA confirmed results obtained from differential display analysis (Fig. 1). The expression level was low when no egg resided in the ESG (3 h after oviposition), increased after the egg entered the ESG (6 h after oviposition), reached a maximum (180% of initial level) at the time of peak eggshell formation (17 h after oviposition), and declined thereafter (23 h after oviposition). Similar results were obtained by PCR analysis (data not shown). To localize alpha 1NKA gene expression, in situ hybridization was performed on ESG biopsies. The epithelium of the ESG consists of two cell types: the pseudostratified epithelium (PE) facing the lumen and the inner glandular epithelium (GE). The alpha 1NKA gene was induced in both cell types, but at different times in the laying cycle. Before the egg entered the ESG, when it was located in the magnum or isthmus (Fig. 2A), or 1 h after the egg entered the ESG (Fig. 2B), no alpha 1NKA gene expression was detected in any epithelial cell. Three hours after the egg had entered the ESG (Fig. 2C), the gene was expressed by most of the PE cells and sporadically by the GE cells. Gradually, more GE cells expressed the alpha 1NKA gene, and 8 h after the egg's entrance into the ESG (Fig. 2D), at the time of rapid shell formation, most of the ESG epithelial cells expressed the alpha 1NKA gene. Four hours later, only the GE cells continued to express a high level of the gene (Fig. 2E), and 1 h before oviposition (Fig. 2F), at the phase of eggshell completion, a massive reduction in gene expression was observed.


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  		Fig. 1.   Northern blot analysis of alpha 1-subunit Na+-K+-ATPase (alpha 1NKA) gene expression during eggshell formation. RNA was taken at different times during the laying cycle (3, 6, 17, and 23 h after oviposition) from 2 different hens. S18 RNA was used as a control of the quantity of loaded RNA in each lane, and the ratio of Na+-K+-ATPase to S18 was plotted.



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  		Fig. 2.   In situ hybridization using the avian alpha 1NKA probe of eggshell gland (ESG) biopsies taken at various times after oviposition: while the egg resides in the isthmus (A) and 1 h (B), 3 h (C), 8 h (D), 12 h (E), and 17 h (F) after the egg enters the ESG. PE, pseudostratified epithelium; GE, glandular epithelium. Arrows, cells expressing the alpha 1NKA gene. Magnification ×50.

Effect of mechanical strain on alpha 1NKA gene expression. We previously demonstrated that mechanical strain induced physiologically or artificially can regulate gene expression in the ESG (33). Insertion of an endotracheal tube resembling the size and shape of an egg into the ESG 3 h after oviposition, at the time when the alpha 1NKA gene is not naturally expressed, induced gene expression (Fig. 3). In contrast to the expression pattern observed during the natural cycle, after application of the mechanical strain the gene was expressed exclusively in the PE cells. In other parts of the oviduct, such as the magnum or isthmus, alpha 1NKA gene expression was induced only when the egg resided there and only by the cells facing the lumen (Fig. 4, A, B, D, and E). These results support our hypothesis that alpha 1NKA gene expression in the PE cells is dependent on mechanical strain. On the other hand, in the colon (Fig. 4, C and F), alpha 1NKA gene expression by the enterocytes did not change during the laying cycle.


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  		Fig. 3.   Effect of mechanical strain on alpha 1NKA gene expression in the ESG. Mechanical strain was applied when the egg did not reside in the ESG and the alpha 1NKA gene was not expressed. A: control, no strain; B: 3 h after mechanical strain induction. Arrows, cells expressing the alpha 1NKA gene. Magnification ×50.



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  		Fig. 4.   Expression of the alpha 1NKA gene in other parts of the oviduct and in the colon. A-C: 12 h after oviposition, the egg resides in the ESG; D and F: 3 h after oviposition, the egg resides in the magnum; E: 5 h after oviposition, the egg resides in the isthmus. Arrows, cells expressing the alpha 1NKA gene. Magnification ×50.

alpha 1NKA gene expression and eggshell calcification. The possibility of an association between alpha 1NKA gene expression and eggshell calcification was evaluated using the carbonic anhydrase inhibitor acetazolamide. Oral administration of this drug 3-4 h after oviposition inhibited eggshell formation, resulting in eggs with soft shells. No changes in alpha 1NKA gene expression were observed in biopsies taken from control and acetazolamide-treated birds 3 h after the egg had entered the ESG, at the time when the alpha 1NKA gene is expressed mainly in the PE cells of the ESG (Fig. 5, A and B). In contrast, a substantial reduction in this gene expression in GE cells was observed 12 h after the egg had entered the ESG, at the time of peak expression in these cells (Fig. 5, C and D).


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  		Fig. 5.   Effect of acetazolamide on alpha 1NKA gene expression. A and C: control, without acetazolamide; B and D: acetazolamide treatment. Sections were taken 3 h (A and B) and 12 h (C and D) after entrance of the egg into the ESG. Arrows, cells expressing the alpha 1NKA gene. Magnification ×50.

Na+-K+-ATPase alpha -subunit isoforms in the ESG. Various isoforms of Na+-K+-ATPase alpha -subunit are known to exist in different proportions in various tissues and to be regulated by different mechanisms. Although in our experiments we used a probe designed to identify the alpha 1-subunit of Na+-K+-ATPase, the possibility of detecting other Na+-K+-ATPase isoforms that share 75% homology to the alpha 1-subunit by this probe could not be ruled out. To exclude the possibility of different isoforms being expressed in the PE and GE cells, we performed a PCR analysis using a set of specific primers, each designed to recognize only one of the three known avian Na+-K+-ATPase isoforms. ESG biopsies were collected at the time of maximal gene expression in PE (6 h after oviposition) and GE (17 h after oviposition) cells. Our results indicate that only the alpha 1-subunit of Na+-K+-ATPase, and not the alpha 2- or the alpha 3-subunit, is expressed in both epithelial cell types of the ESG. Brain, which is known to express all three isoforms, served as a positive control (Fig. 6).


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  		Fig. 6.   Na+-K+-ATPase alpha -isoforms in the ESG. PCR analysis was performed with highly specific primers to 3 known isoforms of avian Na+-K+-ATPase. Only the alpha 1-isoform, not the alpha 2- or alpha 3-isoform, was expressed in the ESG 6 and 17 h after oviposition, at the peak of gene expression in the PE and GE cells, respectively. Brain tissue served as a positive control for all 3 isoforms. MW, molecular weight standard.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The ESG mucosa is a tissue that specializes in active Ca2+ secretion from the plasma to the lumen of the ESG for eggshell formation. The circadian fashion of this massive and rapid Ca2+ transport makes the ESG an ideal model to study the regulation of this process. Ca2+ transport across the ESG mucosa is dependent on at least three different mechanisms: 1) secretion of Ca2+-rich granules, 2) a Ca2+ pump localized in the ESG epithelium, and 3) an ATP-dependent Na+/Ca2+ exchanger, which increases the Ca2+ efflux indirectly by creating an Na+ gradient across the ESG epithelium membranes via Na+-K+-ATPase (36). The association between Ca2+ transport and the concentration of Na+ and K+ has been demonstrated in vitro in mucosa homogenate (36) and in vivo by infusion of an Na+ solution into the ESG cavity (18). Moreover, specific Na+-K+-ATPase inhibitors attenuate Ca2+ secretion as well (46).

Our results indicate that in the ESG the alpha 1NKA gene is markedly induced when eggshell calcification occurs. There was a clear correlation between the level of gene expression and the stage of eggshell formation (Fig. 1). Although the alpha 1NKA gene was expressed in a circadian fashion, a distinct pattern of expression was observed in different epithelial cell types populating the ESG (Fig. 2). As the egg entered the ESG, the alpha 1NKA gene was expressed in the PE cells, and only later was it expressed in the GE cells. As the calcification process progressed toward completion of eggshell formation, the expression of the gene declined, first in the PE cells and only later in the GE cells. These findings raise the possibility of different mechanisms regulating gene expression in the different cell types.

Na+-K+-ATPase is characterized by a complex molecular heterogeneity that results from the expression and differential association of multiple isoforms (4). Different isoforms are expressed in different cell types, in which they contribute to specialized properties (16). The diverse expression of the various isoforms allows distinct cellular responses to different regulators (25, 26, 40, 62). We demonstrated that only the alpha 1-isoform of the enzyme is expressed in both epithelial cell types (Fig. 6), suggesting that the different regulation patterns observed in the PE and GE cells are not due to the existence of different isoforms.

It is known that different cell types express a battery of genes that can reflect their physiological functions. The disparity in the physiological functions between different ESG epithelial cell types has not been clearly defined. Different proteins have diverse localizations in the ESG epithelium. For example, the GE cells express calbindin (13, 59) and Ca2+-ATPase (59, 63), whereas the PE cells express OPN, which is part of the eggshell organic matrix (33, 48). Distinct mechanisms regulating the expression of some of these genes in PE and GE cells have been previously demonstrated (33).

Our results suggest that expression of alpha 1NKA in the ESG is at least partially regulated by Ca2+ flux. The Ca2+ flux required for eggshell formation is partly dependent on the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and this part of the flux is inhibited by the carbonic anhydrase inhibitor acetazolamide. The expression of alpha 1NKA was attenuated in response to acetazolamide-induced changes in Ca2+ flux only in the GE, while its expression in PE cells remained unaltered (Fig. 5). It is interesting to note that the gene encoding calbindin, a Ca2+-binding protein, which is expressed in a circadian manner in GE cells, is also downregulated in response to acetazolamide treatment (33). Laying hens express high levels of 1,25-dihydroxyvitamin D3 and its receptor in the ESG (29, 61). 1,25-Dihydroxyvitamin D3 in chicken induces expression of the beta 1-subunit of Na+-K+-ATPase, suggesting that in the ESG the alpha 1-isoform is probably coupled to the beta 1-isoform. The alpha 1beta 1 isozyme was found to be the most resistant isozyme to changes in intracellular Ca2+ concentration (4). This may explain the existence of only the alpha 1-isoform in the ESG, since the activity of other isoforms would have been inhibited by the high intracellular Ca2+ concentrations during the daily egg cycle.

Recently, we demonstrated that mechanical strain imposed by the resident egg serves as a regulator of OPN gene expression in PE cells of the ESG (33). Mechanical stretching has been reported to increase alpha 1NKA gene expression (5, 50, 56) or the enzyme activity (35, 53, 60). Numerous reports in the literature suggest the involvement of Na+-K+-ATPase in mechanisms underlying diseases connected with mechanical pressure overload, such as hypertension (15, 17), cardiac hypertrophy (7, 10), arteriosclerosis (15, 22), and glomerular diseases (19, 30). It is interesting that only the PE cells responded to physiological and artificial mechanical strain by activating expression of the alpha 1NKA gene, while at the same time these cells did not respond to the Ca2+ flux. The expression of alpha 1NKA is also induced in lumen-facing epithelial cells in other parts of the oviduct (Fig. 4), indicating common features of these epithelial cells in organs with different functions in egg formation.

RhoA, a small GTP-binding protein known to be involved in mechanical strain signal transduction, is also differentially displayed in the ESG at the time of eggshell formation (data not shown). RhoA has been reported to play a role in mechanical stress-induced responses in cardiac myocytes (2) or aortic smooth muscle cells (41) and is implicated as a downstream target of the integrin-dependent signal pathway (11, 21, 23). RhoA has been shown to activate the expression of various transcription factors, such as MyoD (9), c-fos (31, 58), and various other genes via the serum response factor (1, 8, 38, 57). We can only speculate on the involvement of RhoA in the strain-dependent transcriptional activation of the alpha 1-subunit of Na+-K+-ATPase in the ESG. The correlation in the temporal and spatial activation of these two genes, together with the role of RhoA in activating the membrane-associated phospholipase D (6, 20, 24) and inositol lipid kinases (39, 49, 51), which are suggested to affect Na+-K+-ATPase activity (32, 47, 52), supports this hypothesis.

Activation of the alpha 1NKA gene is under the control of multiple protein factors that bind to the positive and negative regulatory regions of the promoter. The level of the overall transcriptional activity appears to depend on the availability of these factors in different tissues in different physiological settings, which would explain the disparity in this gene expression in the different cell types of the ESG and its apparent control by different mechanisms.

In summary, we demonstrate that the alpha 1NKA gene is expressed in different cell types of the ESG and is regulated by various mechanisms, which may reflect the disparity in the physiological role of the cells in the process of eggshell formation.

Perspectives

The avian ESG is a tissue specialized in the massive Ca2+ transport needed for eggshell formation. Being an extracellular process, eggshell formation is governed by 1) proteins responsible for biological processes within the tissue, such as Ca2+ transport and formation of the pH gradient needed for crystal formation and 2) proteins that are secreted from and integrated into the eggshell that regulate the calcification process and become part of the organic shell matrix. At least three interrelated mechanisms regulate the expression of these genes: 1) the mechanical strain imposed locally by the resident egg, 2) circadian rhythm, probably due to systemic hormone secretion, and 3) the Ca2+ flux itself. This occurs in a physiological setting on a daily basis. During the last few years, a search for these regulatory proteins by biochemical and molecular approaches was made, and some general rules have started to emerge. 1) Part of the organic matrix of the shell consists of proteoglycans that probably contribute to the biochemical properties of the mature product. 2) Some, but not all, of the proteins known to play a role in bone formation were found to be involved in shell formation as well. This is of major interest, since bone and eggshell consist of hydroxyapatite and calcium carbonate, respectively. 3) Genes coding for proteins involved in ion transport, such as calbindin and Na+-K+-ATPase, are upregulated. In the case of the Na+-K+-ATPase, different mechanisms of the regulation of a specific subunit were identified in various cell populations, suggesting another level of control. Together, this unique system can be used for the elucidation of fundamental physiological questions.


    FOOTNOTES

Address for reprint requests and other correspondence: M. Pines, Institute of Animal Sciences, ARO, The Volcani Center, Bet Dagan 50250, Israel (E-mail: pines{at}agri.huji.ac.il).

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. Section 1734 solely to indicate this fact.

Received 8 February 2001; accepted in final form 21 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aicher, WK, Dinkel A, Grimbacher B, Haas C, Seydlitz-Kurzbach EV, Peter HH, and Eibel H. Serum response elements activate and cAMP responsive elements inhibit expression of transcription factor Egr-1 in synovial fibroblasts of rheumatoid arthritis patients. Int Immunol 11: 47-61, 1999[Abstract/Free Full Text].

2.   Aikawa, R, Komuro I, Yamazaki T, Zou Y, Kudoh S, Zhu W, Kadowaki T, and Yazaki Y. Rho family small G proteins play critical roles in mechanical stress-induced hypertrophic responses in cardiac myocytes. Circ Res 84: 458-466, 1999[Abstract/Free Full Text].

3.   Bar, A, Striem S, Vax E, Talpaz H, and Hurwitz S. Regulation of calbindin mRNA and calbindin turnover in intestine and shell gland of the chicken. Am J Physiol Regulatory Integrative Comp Physiol 262: R800-R805, 1992[Abstract/Free Full Text].

4.   Blanco, G, and Mercer RW. Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function. Am J Physiol Renal Physiol 275: F633-F650, 1998[Abstract/Free Full Text].

5.   Book, CB, Wilson RP, and Ng YC. Cardiac hypertrophy in the ferret increases expression of the Na+-K+-ATPase alpha 1- but not alpha 3-isoform. Am J Physiol Heart Circ Physiol 266: H1221-H1227, 1994[Abstract/Free Full Text].

6.   Bowman, EP, Uhlinger DJ, and Lambeth JD. Neutrophil phospholipase D is activated by a membrane-associated Rho family small-molecular-weight GTP-binding protein. J Biol Chem 268: 21509-21512, 1993[Abstract/Free Full Text].

7.   Bundgaard, H, and Kjeldsen K. Human myocardial Na,K-ATPase concentration in heart failure. Mol Cell Biochem 163-164: 277-283, 1996.

8.   Camoretti-Mercado, B, Liu HW, Halayko AJ, Forsythe SM, Kyle JW, Li B, Fu Y, McConville J, Kogut P, Vieira JE, Patel NM, Hershenson MB, Fuchs E, Sinha S, Miano JM, Parmacek MS, Burkhardt JK, and Solway J. Physiological control of smooth muscle-specific gene expression through regulated nuclear translocation of serum response factor. J Biol Chem 275: 30387-30393, 2000[Abstract/Free Full Text].

9.   Carnac, G, Primig M, Kitzmann M, Chafey P, Tuil D, Lamb N, and Fernandez A. RhoA GTPase and serum response factor control selectively the expression of MyoD without affecting Myf5 in mouse myoblasts. Mol Biol Cell 9: 1891-1902, 1998[Abstract/Free Full Text].

10.   Charlemagne, D, and Swynghedauw B. Myocardial phenotypic changes in Na+,K+-ATPase in left ventricular hypertrophy: pharmacological consequences. Eur Heart J 16, SupplC: 20-23, 1995.

11.   Clark, EA, King WG, Brugge JS, Symons M, and Hynes RO. Integrin-mediated signals regulated by members of the rho family of GTPases. J Cell Biol 142: 573-586, 1998[Abstract/Free Full Text].

12.   Corradino, RA. Calbindin D28K regulation in precociously matured chick egg shell gland in vitro. Gen Comp Endocrinol 91: 158-166, 1993[ISI][Medline].

13.   Corradino, RA, Smith CA, Krook LP, and Fullmer CS. Tissue-specific regulation of shell gland calbindin D28K biosynthesis by estradiol in precociously matured, vitamin D-depleted chick. Endocrinology 132: 193-198, 1993[Abstract].

14.   Creger, CR, Phillips H, and Scott JJ. Formation of an eggshell. Poult Sci 55: 1717-1723, 1976.

15.   Davies, MJ. Hypertension and atherosclerotic (ischaemic) heart disease. J Hum Hypertens 5, Suppl1: 23-29, 1991.

16.   Decollogne, S, Bertrand IB, Ascensio M, Drubaix I, and Lelievre LG. Na+,K+-ATPase and Na+/Ca2+ exchange isoforms: physiological and physiopathological relevance. J Cardiovasc Pharmacol 22, Suppl2: S96-S98, 1993.

17.   Delcayre, C, and Swynghedauw B. Biological adaptation and dysadaptation of the heart to chronic arterial hypertension. J Hypertens Suppl 9: S23-S29, 1991[Medline].

18.   Eastin, WC, Jr, and Spaziani E. On the mechanism of calcium secretion in the avian shell gland (uterus). Biol Reprod 19: 505-518, 1978[Abstract].

19.   Eiam-Ong, S, Laski ME, and Kurtzman NA. Diseases of renal adenosine triphosphatase. Am J Med Sci 309: 13-25, 1995[ISI][Medline].

20.   Fensome, A, Whatmore J, Morgan C, Jones D, and Cockcroft S. ADP-ribosylation factor and Rho proteins mediate fMLP-dependent activation of phospholipase D in human neutrophils. J Biol Chem 273: 13157-13164, 1998[Abstract/Free Full Text].

21.   Fox, JE. On the role of calpain and Rho proteins in regulating integrin-induced signaling. Thromb Haemost 82: 385-391, 1999[ISI][Medline].

22.   Frangos, SG, Gahtan V, and Sumpio B. Localization of atherosclerosis: role of hemodynamics. Arch Surg 134: 1142-1149, 1999[Abstract/Free Full Text].

23.   Hotchin, NA, and Hall A. The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases. J Cell Biol 131: 1857-1865, 1995[Abstract/Free Full Text].

24.   Houle, MG, and Bourgoin S. Small GTPase-regulated phospholipase D in granulocytes. Biochem Cell Biol 74: 459-467, 1996[ISI][Medline].

25.   Huang, L, Kometiani P, and Xie Z. Differential regulation of Na/K-ATPase alpha -subunit isoform gene expressions in cardiac myocytes by ouabain and other hypertrophic stimuli. J Mol Cell Cardiol 29: 3157-3167, 1997[ISI][Medline].

26.   Gao, J, Wymore R, Wymore RT, Wang Y, McKinnon D, Dixon JE, Mathias RT, Cohen IS, and Baldo GJ. Isoform-specific regulation of the sodium pump by alpha - and beta -adrenergic agonists in the guinea-pig ventricle. J Physiol (Lond) 516: 377-383, 1999[Abstract/Free Full Text].

27.   Glynn, IM. The Na,K-transporting adenosine triphosphatase. In: The Enzymes of Biological Membranes, edited by Martonosi A. New York: Plenum, 1992, vol. 3, p. 35-114.

28.   Glynn, IM. All hands to the sodium pump. J Physiol (Lond) 462: 1-30, 1993[Free Full Text].

29.   Ieda, T, Saito N, Ono T, and Shimada K. Effects of presence of an egg and calcium deposition in the shell gland on levels of messenger ribonucleic acid of CaBP-D28K and of vitamin D3 receptor in the shell gland of the laying hen. Gen Comp Endocrinol 99: 145-151, 1995[ISI][Medline].

30.   Ingram, AJ, and Scholey JW. Stress-responsive signal transduction mechanisms in glomerular cells. Curr Opin Nephrol Hypertens 9: 49-55, 2000[ISI][Medline].

31.   Kim, BC, Lim CJ, and Kim JH. Arachidonic acid, a principal product of Rac-activated phospholipase A2, stimulates c-fos serum response element via Rho-dependent mechanism. FEBS Lett 415: 325-328, 1997[ISI][Medline].

32.   Lattimer, SA, Sima AA, and Greene DA. In vitro correction of impaired Na+-K+-ATPase in diabetic nerve by protein kinase C agonists. Am J Physiol Endocrinol Metab 256: E264-E269, 1989[Abstract/Free Full Text].

33.   Lavelin, I, Yarden N, Ben-Bassat S, Bar A, and Pines M. Regulation of osteopontin gene expression during egg shell formation in the laying hen by mechanical strain. Matrix Biol 17: 615-623, 1998[ISI][Medline].

34.   Lingrel, JB, and Kuntzweiler T. Na+,K+-ATPase. J Biol Chem 269: 19659-19662, 1994[Free Full Text].

35.   Liu, X, Hymel LJ, and Songu-Mize E. Role of Na+ and Ca2+ in stretch-induced Na+-K+-ATPase alpha -subunit regulation in aortic smooth muscle cells. Am J Physiol Heart Circ Physiol 274: H83-H89, 1998[Abstract/Free Full Text].

36.   Lundholm, CE. Relation between Ca metabolism and ATPase activities in the subcellular fractions from duck eggshell gland mucosa after DDE administration during different stages of eggshell formation. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 82: 1-16, 1985.

37.   Mercer, RW. Structure of the Na,K-ATPase. Int Rev Cytol 137C: 139-168, 1993.

38.   Miano, JM, Carlson MJ, Spencer JA, and Misra RP. Serum response factor-dependent regulation of the smooth muscle calponin gene. J Biol Chem 275: 9814-9822, 2000[Abstract/Free Full Text].

39.   Narumiya, S. The small GTPase Rho: cellular functions and signal transduction. J Biochem (Tokyo) 120: 215-228, 1996[Abstract/Free Full Text].

40.   Nishi, A, Fisone G, Snyder GL, Dulubova I, Aperia A, Nairn AC, and Greengard P. Regulation of Na+,K+-ATPase isoforms in rat neostriatum by dopamine and protein kinase C. J Neurochem 73: 1492-1501, 1999[ISI][Medline].

41.   Numaguchi, K, Eguchi S, Yamakawa T, Motley ED, and Inagami T. Mechanotransduction of rat aortic vascular smooth muscle cells requires RhoA and intact actin filaments. Circ Res 85: 5-11, 1999[Abstract/Free Full Text].

42.   Nys, Y. Regulation of plasma 1,25(OH)2D3, of osteocalcin and of intestinal and uterine calbindin in hens. In: Avian Endocrinology, edited by Sharp PJ. Bristol, UK: Journal of Endocrinology Limited, 1993, p. 345-357.

43.   Nys, Y, Baker K, and Lawson DE. Estrogen and a calcium flux-dependent factor modulate the calbindin gene expression in the uterus of laying hens. Gen Comp Endocrinol 87: 87-94, 1992[ISI][Medline].

44.   Nys, Y, Mayel-Afshar S, Bouillon R, Van Baelen H, and Lawson DE. Increases in calbindin D 28K mRNA in the uterus of the domestic fowl induced by sexual maturity and shell formation. Gen Comp Endocrinol 76: 322-329, 1989[ISI][Medline].

45.   Pearson, TW, and Goldner AM. Calcium transport across avian uterus. I. Effects of electrolyte substitution. Am J Physiol 225: 1508-1512, 1973.

46.   Pearson, TW, and Goldner AM. Calcium transport across avian uterus. II. Effects of inhibitors and nitrogen. Am J Physiol 227: 465-468, 1974.

47.   Pierce, GN, and Panagia V. Role of phosphatidylinositol in cardiac sarcolemmal membrane sodium-calcium exchange. J Biol Chem 264: 15344-15350, 1989[Abstract/Free Full Text].

48.   Pines, M, Knopov V, and Bar A. Involvement of osteopontin in egg shell formation in the laying chicken. Matrix Biol 14: 765-771, 1995[ISI][Medline].

49.   Ren, XD, and Schwartz MA. Regulation of inositol lipid kinases by Rho and Rac. Curr Opin Genet Dev 8: 63-67, 1998[ISI][Medline].

50.   Ruiz-Opazo, N, Xiang XH, and Herrera VL. Pressure-overload deinduction of human alpha 2 Na,K-ATPase gene expression in transgenic rats. Hypertension 29: 606-612, 1997[Abstract/Free Full Text].

51.   Schmidt, M, Rumenapp U, Keller J, Lohmann B, and Jakobs KH. Regulation of phospholipase C and D activities by small molecular weight G proteins and muscarinic receptors. Life Sci 60: 1093-1100, 1997[ISI][Medline].

52.   Simpson, CM, and Hawthorne JN. Reduced Na+ + K+- ATPase activity in peripheral nerve of streptozotocin-diabetic rats: a role for protein kinase C? Diabetologia 31: 297-303, 1988[ISI][Medline].

53.   Songu-Mize, E, Liu X, and Hymel LJ. Effect of mechanical strain on expression of Na+,K+-ATPase alpha -subunits in rat aortic smooth muscle cells. Am J Med Sci 316: 196-199, 1998[ISI][Medline].

54.   Stemberger, BH, Mueller WJ, and Leach RM. Microscopic study of the initial stage of eggshell calcification. Poult Sci 56: 537-543, 1977.

55.   Striem, S, and Bar A. Modulation of quail intestinal and egg shell gland calbindin (Mr 28,000) gene expression by vitamin D3, 1,25-dihydroxyvitamin D3 and egg laying. Mol Cell Endocrinol 75: 169-177, 1991[ISI][Medline].

56.   Sznajder, JI, Ridge KM, Harris ZL, Olivera W, Curiel C, and Rutschman DH. Alveolar type II cell Na,K-ATPase is upregulated during mechanical ventilation-induced pulmonary edema. Chest 105, Suppl3: 116S-117S, 1994.

57.   Treisman, R, Alberts AS, and Sahai E. Regulation of SRF activity by Rho family GTPases. Cold Spring Harb Symp Quant Biol 63: 643-651, 1998[ISI][Medline].

58.   Ueyama, T, Sakoda T, Kawashima S, Hiraoka E, Hirata K, Akita H, and Yokoyama M. Activated RhoA stimulates c-fos gene expression in myocardial cells. Circ Res 81: 672-678, 1997[Abstract/Free Full Text].

59.   Wasserman, RH, Smith CA, Smith CM, Brindak ME, Fullmer CS, Krook L, Penniston JT, and Kumar R. Immunohistochemical localization of a calcium pump and calbindin-D28k in the oviduct of the laying hen. Histochemistry 96: 413-418, 1991[ISI][Medline].

60.   Waters, CM, Ridge KM, Sunio G, Venetsanou K, and Sznajder JI. Mechanical stretching of alveolar epithelial cells increases Na+-K+-ATPase activity. J Appl Physiol 87: 715-721, 1999[Abstract/Free Full Text].

61.   Wu, JC, Smith MW, Turvey A, Keable SJ, and Colston KW. Differential regulation of vitamin D receptor and intestinal calcium transport occurring during sexual maturation in the fowl (Gallus domesticus). Comp Biochem Physiol A Physiol 109: 713-720, 1994[Medline].

62.   Yalcin, Y, Carman D, Shao Y, Ismail-Beigi F, Klein I, and Ojamaa K. Regulation of Na/K-ATPase gene expression by thyroid hormone and hyperkalemia in the heart. Thyroid 9: 53-59, 1999[ISI][Medline].

63.   Yamamoto, T, Ozawa H, and Nagai H. Histochemical studies of Ca-ATPase, succinate and NAD+-dependent isocitrate dehydrogenases in the shell gland of laying Japanese quails: with special reference to calcium-transporting cells. Histochemistry 83: 221-226, 1985[ISI][Medline].

Am J Physiol Regul Integr Comp Physiol 281(4):R1169-R1176
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