Calcium Homeostasis and Vitamin D Metabolism and Expression in Strongly Calcifying Laying Birds

Preprint Review Calcium homeostasis and vitamin D metabolism and expression in strongly calcifying laying birds Arie Bar1 Institute of Animal Science, ARO, The Volcani Ctr., Bet Dagan, Israel

Running head: Vitamin D in laying birds

Published in: Comparative Biochemistry and Physiology, Part A: Molecular & Integrative Physiology, (2008) 151: 477-490. For the printed version please link to: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi? cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18682298

1 Retired; Institute of Animal Science, ARO, the Volcani Ctr., Bet Dagan 50250, Israel; E-mail: [email protected] 2

ABSTRACT Egg laying and shell calcification impose severe extra demands on ionic calcium (Ca2+) homeostasis; especially in birds characterized by their long clutches (series of eggs laid sequentially before a “pause day”). These demands induce vitamin D metabolism and expression. The metabolism of vitamin D is also altered indirectly, by other processes associated with increased demands for calcium, such as growth, bone formation and egg production. A series of intestinal, renal or bone proteins are consequently expressed in the target organs via mechanisms involving a vitamin D receptor. Some of these proteins (carbonic anhydrase, calbindin and calcium-ATPase) are also found in the uterus (eggshell gland) or are believed to be involved in calcium transport in the intestine or kidney (calcium channels). The present review deals with vitamin D metabolism and the expression of the above-mentioned proteins in birds, with special attention to the strongly calcifying laying bird.

Keywords: calbindin, calcium, carbonic anhydrase, eggshell gland (uterus), intestine, vitamin D (cholecalciferol) 3

Contents 1. Introduction...... 4 2. Calcium in avian reproduction...... 4 2.1. Homeostasis: 4 2.2. Medullary bone 8 3. Vitamin D metabolism and its regulation in birds...... 8 3.1. Non-laying vertebrates 9 3.2. Laying birds 12 4. Vitamin D expression...... 15 5. Vitamin D-dependent proteins...... 16 5.1. Vitamin D receptor 16 5.2. Calbindins 17 5.3. Plasma membrane calcium-ATPase (Ca2+ATPase or PMCA) 23 5.4. Epithelial calcium channels (TRPVs) 24 5.5. Carbonic anhydrase 25 6. Vitamin D-controlled transcellular transport of Ca2+...... 26 Concluding remarks...... 27 Acknowledgments...... 28 References...... 29 4

1. Intoduction Birds that lay long clutches (series of eggs laid sequentially before a “pause day”), among them the high-producing Gallus domesticus (domestic hen) and Coturnix coturnix japonica (Japanese quail) transfer about 10% of their total body calcium daily. This imposes severe demands on ionic calcium (Ca2+) homeostasis, and activates very efficient mechanisms for Ca2+ transfer from food to the eggshell. Two major calcium-regulating hormones are involved in these phenomena: the hormonally active vitamin D metabolite, 1,25-dihydroxy vitamin D3

(1,25(OH)2D3), and Parathyroid Hormone (PTH). Whereas PTH acts rapidly and directly – mostly on the kidney and bone – vitamin D acts more slowly, at the intestinal level also. Neither of these hormones has yet been found to affect eggshell gland (ESG; uterus) calcium transport directly, but many of the proteins believed to be vitamin D dependent are present and are modified in the ESG during egg laying by birds with long clutches.

The present review focuses on the metabolism of the hormonal form of 1,25(OH)2D3 in laying birds; and on the expression of some out the many vitamin D-induced genes believed to be associated with Ca2+ transport in the intestine and ESG, rather than on the mechanisms of Ca2+ transport in the laying bird. These mechanisms will be discussed separately (Bar, in preparation).

2. Calcium in avian reproduction 2.1. Homeostasis: In order to maintain their extra-cellular Ca2+ concentration, land vertebrates implement homeostasis through a complex feedback mechanism that involves three major controlled systems, i.e., intestine, kidney and bone, and three major calcium-regulating hormones, i.e., PTH (reviewed in (Dacke 2000; Akerstrom et al. 2005; Potts 2005; Guerreiro et al. 2007; Potts et al. 2007)2, calcitonin, (reviewed in (Dacke 2000; Findlay et al. 2004; Huang et al.

2006) and 1,25-dihydroxycholecalciferol (1,25-dihydroxyvitamin D3; 1,25(OH)2D3) which is

2+ the hormonal form of cholecalciferol (vitamin D3; see 3). Changes (errors) in the plasma Ca concentration are detected by Ca2+-sensing receptors (reviewed in (Miller 1992; Hurwitz 1996; Sasayama 1999; Ramasamy 2006). The controlled systems respond to detected errors in plasma Ca2+ concentration by adjusting the transport of Ca2+ into and out of the extra- cellular pool (Fig. 1).

2 In order to limit the review length, only few of the relevant reviews or original articles were cited. In most cases the most recent reviews and the earlier original papers were cited. In some cases were cited other (more relevant or more comprehensive) publications. 5

Fig. 1. Ca2+ homeostasis in the laying bird. Closed black arrows, Ca2+ flow; closed gray arrows, hormonal regulation; dotted black arrows, plasma Ca2+ regulation; dashed gray arrows, uncertain (controversial results) 2+ hormonal regulation (dCa/dt, the change in plasma Ca ; PTH, parathyroid hormone; 1,25D3, 1,25(OH)2D3). Values given in parentheses are g calcium per day (Hurwitz et al. 1969). More recent studies indicated that the modern breeds/lines of laying domestic hens eat up to 5 g calcium per day and are able to deposit into the shell up to 2.7 g calcium per a single egg cycle (practically during the 2nd half of the cycle). However, due to animal welfare restrictions/regulations, the recent publications are not distinguishing between fecal and urinary calcium excretion (as it requires a surgery procedure). 6

In birds, especially those with long clutches, egg laying and shell calcification introduces an additional Ca2+ pathway: its withdrawal through the uterus (egg shell gland, ESG). This imposes severe extra demands on Ca2+ homeostasis, since shell formation requires several times as much calcium as exists in the extra-cellular pool. These demands arise firstly through the initiation of gonadal activity during maturation prior to egg laying, to support new medullary bone (MB) formation and to saturate the estrogen-dependent plasma-calcium- binding proteins formed in the liver (reviewed in (Griffin 1992; Walzem 1996; Walzem et al. 1999)), and then from the enhanced requirements for shell Ca2+ during the egg-laying phase. The deposition of shell calcium appears not to be controlled by the three calcium- regulating hormones, or, at least, to be controlled differently from Ca2+ transport in the intestine, bone and kidney (Table 1; Bar et al. 1973b; Bar et al. 1975a; Bar et al. 1978b; Bar et al. 1984b; Bar et al. 1990b; Bar et al. 1992a; Bar et al. 1992c; Nys et al. 1989; Striem 1990). The intestinal lumen becomes almost completely empty of calcium 4 to 5 h after the end of the feeding stage (or when the light is turned off in birds with free access to food) (Hurwitz et al. 1966), whereas the intensive shell calcification in the commercial hen occurs between 9 and 22 h after ovulation, i.e., -1 to 12 h after lights-out, and consequently a major proportion of the shell Ca2+ is deposited while the intestine lacks adequate dietary calcium. Therefore, two mechanisms are enhanced: (a) enhancement of net absorption of Ca2+ during the dark period, especially early in that period when feed is still present in the gut (Hurwitz et al. 1965; Hurwitz et al. 1973; Bar et al. 1976b); and (b) the activation of an efficient process of bone resorption, mostly of the “medullary bone” (MB; details in the next paragraph.). Both mechanisms act during the period of shell calcification that occurs overnight. Restoration of the circadian bone loss of Ca2+ in birds that lay long sequential clutches occurs during the subsequent daylight period, when the intestinal absorption of Ca2+ is enabled by the renewal of calcium intake. 7

Table 1. Differential regulation of avian calbindin: Effects of selected physiological and nutritional alterations ______Cause/tissue Intestine Kidney ESG References1 ______

VVitamin D derivatives in vitamin-D-deficient2 +++3 ++ =/+4 (Wasserman et al. 1966; Corradino et al. 1968; Taylor et al. 1972; Striem et al. 1991) 1-hydroxylated derivatives in vitamin-D-fed +++ = = (Bar et al. 1973c; Bar et al. 1975b; Bar et al. 1976b) Dietary Ca2+ restriction in vitamin-D-fed +++ =/- =/- (Wasserman et al. 1968; Bar et al. 1972a; Bar et al. 1973b; Bar et al. 1975b; Bar et al. 1978b) Dietary P restriction in vitamin-D-fed +++ +++ = (Morrissey et al. 1971; Bar et al. 1973c; Bar et al. 1975b; Bar et al. 1984a) Dietary Ca2+ restriction in 1-hydroxylated-D-fed = = ND5 (Bar et al. 1973c; Bar et al. 1975b) Dietary P restriction in 1-hydroxylated-D-fed +++ +++ ND (Bar et al. 1973c; Bar et al. 1975b) High dietary Ca2+ in D-fed -- + ND (Bar et al. 1990a) Growth ++ = (Bar et al. 1981a) Maturation or gonadal hormones ++ = =/+ (Bar et al. 1972a; Bar et al. 1973b; Montecuccoli et al. 1977b; Bar et al. 1978b; Bar et al. 1979a; Navickis et al. 1979a; Navickis et al. 1979b) Laying +++ = +++ (Corradino et al. 1968; Wasserman et al. 1968; Bar et al. 1972a; Bar et al. 1973b; Bar et al. 1978b) Shell calcification (on protein synthesis) = ND = (Bar et al. 1975a) Shell calcification (on mRNA synthesis) = ND +++ (Nys et al. 1989; Striem et al. 1991; Bar et al. 1992a; Nys et al. 1992a) ______1 The table tries to bring together most earlier published quantitative or semi-quantitative comparisons, as well as supporting evidence published later by other research teams. Many, but not all, other studies are mentioned in the text and in the References list. Few of the very early studies used the chelex assay, but were confirmed later with immunoassays or Western analysis. 2 Not laying. 3 The regulative response varied between non (=) to very strong (+++) or negative (-); ND, not detected; 4 Varied in accordance with laying rate. 5 Not determined. 8

2.2. Medullary bone: The unique MB is formed in the marrow cavities of the long bones of mature female birds (reviewed in (Dacke et al. 1993b; Gay 1996; Sugiyama et al. 2001; Beck et al. 2004; Whitehead 2004)). The MB accumulates rapidly during sexual maturation and the onset of egg production, and slow accumulation of its components continues throughout the laying period. The MB does not appear to have a structural function, but it serves as a labile source of Ca2+ for shell deposition. Such a labile source enables shell calcification in birds with long clutches or in birds that calcify eggs during the dark hours in the absence of an adequate intestinal supply of calcium (see 2.1). The formation and maintenance of MB require the combined effects of estrogen and androgen (Bloom et al. 1942); in addition, its mineralization requires the intestinal supply of

2+ Ca and the active metabolite of vitamin D: 1,25(OH)2D3 (Kaetzel et al. 1984; Newbrey et al. 1992; Newman et al. 1999). During the 24- to 25.5-h cycle of egg formation in chicks and quails, MB is formed and resorbed by the osteoblasts and osteoclasts, respectively. The changes in the MB are manifested in its degree of calcification, rather than its volume (reviewed in (Dacke et al. 1993b)). Prior to the entry of an egg into the ESG, MB calcification/formation by osteoblasts is induced in response to estrogen secretion by mature follicles; its binding to specific receptors (reviewed in ( Sugiyama et al. 2001) occur mostly onthe osteoblasts plasma membrane (Armen et al. 2000). During shell calcification, the plasma Ca2+ concentration declines, which induces secretion of PTH (Dacke 1976; van de Velde et al. 1984; Singh et al. 1986) and its binding to specific receptors (Yasuoka et al. 1996) (reviewed in (Sugiyama et al. 2001)), and this, in turn, prompts the resorption by the osteoclasts.

Whereas the roles of gonadal hormones and 1,25(OH)2D3 in MB formation, and of PTH in MB resorption are well established, the involvement of calcitonin, which is secreted by the ultimobranchial glands (Eliam et al. 1988; Dacke et al. 1993a; Sugiyama et al. 2001), and of Ca2+-sensing receptors (Zaidi et al. 1996) in MB remodeling are not yet certain.

3. Vitamin D metabolism and its regulation in birds

The active metabolite of vitamin D3, i.e., 1,25(OH)2D3 was first known to affect the classical calcium-transporting organs such as the intestine, kidney and bone, as well as some non-classical organs and functions such as cell growth, immune response and apoptosis. Among the factors modulating Ca2+ absorption in birds, vitamin D appears to be the most important. Many other factors, such as dietary content of Ca2+ and/or phosphorus (P), 9

maturation and gonadal activity, egg laying and shell calcification, appear to modulate Ca2+ absorption, mainly through their effects on vitamin D metabolism and expression.

3.1. Non-Laying Vertebrates Metabolism: The metabolism and expression of vitamin D in non-laying land vertebrates have been extensively reviewed elsewhere (Bouillon et al. 1997; Henry 1997; Pike 1997; Haussler et al. 1998; Jones et al. 1998; Brown et al. 1999; Holick 2003; DeLuca 2004; Dusso et al. 2005; Norman 2006). Vitamin D metabolism in birds is basically similar to that in mammals, but was less extensively reviewed (Hurwitz 1992; Norman 1995; Soares et al. 1995; Wasserman 1997; Whitehead 1998; Edwards 2000). Briefly (Fig. 2): in birds (and in a few New World primates) the bioactivity of vitamin D3 is higher than that of ergocalciferol

(vitamin D2), because the affinity of avian plasma vitamin D binding proteins (DBD) is markedly lower for vitamin D2 than for D3 derivatives, whereas the affinity of mammalian

DBD (DeLuca et al. 1988) is similar for both. Vitamin D3, either from a dietary source or synthesized in the skin from 7-dehydroxycholesterol in response to ultraviolet irradiation and skin temperature is bioactivated, first in the liver and than in the kidney. This bioactivation (reviewed in (Omdahl et al. 2002; Ohyama et al. 2004; Prosser et al. 2004; Dusso et al. 2005)) involves the participation of three or more cytochrome P450 (CYP) isoforms. Vitamin D3 is first 25-hydroxylated in the liver microsomes and mitochondria to form 25-hydroxyvitamin D (25OHD3), which is the major circulating metabolite. The microsomal 25-hydroxylase in the liver (CYPR1) is capable of functioning at the physiological substrate concentration. Mitochondrial 25-hydroxylation appears to use another P450 isoform (out of at least six isoforms that exhibit vitamin D 25-hydroxylation activities). It appears to be species specific, and it also appears to use a non-specific pathway associated with vitamin D intoxication. Similarly to other vitamin D metabolites, 25OHD3 is mostly bound to DBD and appears to be slightly more active than vitamin D3 itself, apparently because of its better intestinal absorption (Bar et al. 1980). 10

Fig. 2. Vitamin D metabolism and regulation in the laying bird (EST, estrogens; GH, growth hormone; Pl-Ca 2+, plasma Ca2+; Pl-P, plasma P-; PTH, parathyroid hormone). 11

Activation of 25OHD3 synthesis occurs in the kidney mitochondria, where it is further

1-hydroxylated by the 25-hydroxy-vitamin-D-1-hydroxylase (1-hydroxylase; CYP27B1), to form the hormonal form of vitamin D3, 1,25(OH)2D3. The kidney also expresses, a high activity of vitamin D-derived 24-hydroxylase (24-hydroxylase; CYP24A1), which is, most likely, responsible for inactivation of vitamin D derivatives. The renal vitamin D 24- hydroxylase catalyzes side-chain cleavage and inactivation of 25OHD3 and 1,25(OH)2D3. Although the major site of activity of both 24- and 1-hydroxylases is the kidney, both are found in other tissues also, including the mammalian placenta, intestine, bone and skin.

Regulation of 25OHD3 and 1,25(OH)2D3 formation and degradation: 25-hydroxylation in the liver is not strictly feedback regulated; it mainly reflects substrate availability. Therefore, the level of plasma 25OHD3 appears to be a good indicator of dietary vitamin D3 content or of vitamin D status.

Renal 1-hydroxylation of 25OHD3 and the formation of 1,25(OH)2D3 is fine-regulated by plasma Ca2+ and PTH. Contradictory opinions have been expressed regarding the possible direct role(s) in 1,25(OH)2D3 formation of gonadal (Tanaka et al. 1976; Baksi et al. 1978; Bar et al. 1979a; Nys et al. 1984c; Sommerville et al. 1989; Striem 1990) or other hormones such as prolactin or growth hormone (Spanos et al. 1976) or dietary phosphorus (P) restriction (Henry et al. 1974; Ribovich et al. 1975; Montecuccoli et al. 1977b; Bar et al. 1981a), but their involvement is generally accepted. Dietary Ca2+ restrictions and rapid growth stimulate 1-hydroxylation, most likely through increased PTH secretion. In birds, but not in mammals, dietary P restriction does not induce renal 1-hydroxylation (Henry et al. 1974; Montecuccoli et al. 1977b), although the contents of the hormone in the plasma and target organs are increased (Edelstein et al. 1975; Hunziker et al. 1982; Rosenberg et al. 1986). This could be attributed either to an elevation in vitamin D receptor (VDR) (see 5.1) content or affinity, or possibly to retarded clearance of 1,25(OH)2D3 in P-restricted birds. Whereas there is some evidence to support the first explanation (Meyer et al. 1992), the clearance of 1,25(OH)2D3 in

P-deficient birds has not yet been determined. Age and high levels of circulating 1,25(OH)2D3 moderate the responses of birds (Bar et al. 1981a) and mammals (Armbrecht et al. 1980a; Armbrecht et al. 1980b) to dietary minerals restriction or to growth. Acidosis in the chick is associated with a decreased 1-hydroxykase activity and/or plasma

1,25(OH)2D3 contents (Booth et al. 1977; Sauveur et al. 1977; Cunningham et al. 1987). Similar findings were obtained in mammals, including rats, cats and humans (Reddy et al. 1982; Cunningham et al. 1984; Ching et al. 1989). In humans, these effect are observed in 12

acute, but not in chronic acidosis (Cunningham et al. 1984). This reduction is attributed to an indirect effect of acidosis, manifested through plasma Ca2+ or PTH, but not plasma H+ (Bushinsky et al. 1985; Ro et al. 1990). The 24-hydroxylase is regulated in a reciprocal manner to 1-hydroxylase. The gene encoding vitamin D 24-hydroxylase contains at least two distinct vitamin D-responsive elements (VDREs) (see 5.1) that mediate the effects of 1,25(OH)2D3 via its receptor, on transcription.

Although the present review deals mainly with 1,25(OH)2D3, the reader should be aware of the biological activities of other 25OH-metabolites, among which is 24,25(OH)2D3. This metabolite was found to be involved in: bone remodeling in mammals and birds (Henry et al. 1976; Ornoy et al. 1978), bone fracture healing (Lidor et al. 1990) and egg hatchability ((Henry et al. 1978) reviewed in (Norman et al. 2002)). However, these metabolites were not yet shown to have a significant role in Ca2+ transport in laying birds (Grunder et al. 1990). On the other hand, some authors (Hart et al. 1984; Harrison et al. 1986) doubted some of the above findings.

3.2. Laying Birds Basically, the metabolism of vitamin D and its regulation are similar in mammals and in both non-laying and laying birds. However, sexual maturation and the subsequent onset of egg-laying challenge the homeostasis of Ca2+ (see 2.1) and, consequently, modulate vitamin D metabolism and expression.

3.2.1. Regulation of 1,25(OH)2D3 formation: The renal formation of 1,25(OH)2D3 and its concentration in the plasma are markedly increased in the laying bird (Montecuccoli et al. 1977b; Baksi et al. 1978; Bar et al. 1981b). First, the combined elevation of plasma estrogens and androgens that occurs during maturation stimulates the renal formation of 1,25(OH)2D3, either directly (Tanaka et al. 1976; Castilo et al. 1979), or indirectly through the formation of MB and the binding of calcium to plasma proteins (Bar et al. 1979a). Later, the onset of laying further stimulates 1,25(OH)2D3 synthesis (Kenny 1976; Spanos et al. 1976; Montecuccoli et al. 1977b; Bar et al. 1981b; Nys et al. 1992a), most likely as a result of the increased need for Ca2+ for the shell (Bar et al. 1978a; Nys et al. 1986b; Nys et al. 1992c). The latter is associated with a decrease in plasma Ca2+ and an increase in plasma PTH (van de Velde et al. 1984; Nys et al. 1986a; Singh et al. 1986; Frost et al. 1990; Ieda et al. 2000; Sugiyama et al. 2001; Goto et al. 2002b). The changes in plasma Ca2+ and PTH are considered by some researchers to stimulate the renal-1-hydroxylation during the period of 13

shell calcification (see next paragraph). Further stimulation of 1,25(OH)2D3 synthesis is induced by dietary calcium restriction (even if due to brief food withdrawal or overnight starvation), laying rate (Bar et al, unpublished results), and/or during the “pause” day(s) at the end of a clutch (Montecuccoli et al. 1977b; Sedrani et al. 1977; Bar et al. 1984b). Age slows down renal-1-hydroxylation, but the mechanism of this effect is uncertain: some data suggest that age reduces the basal metabolism of vitamin D (Abe et al. 1982; Joyner et al. 1987) whereas other findings suggest that it mainly affects the adaptive mechanism, in accordance with the fluctuating demand for dietary Ca (Bar et al. 1987). Similar effects of age on the adaptation of vitamin D metabolism and expression were demonstrated also in mammals (Armbrecht et al. 1980b; Armbrecht et al. 1999). The effect of the egg cycle on the renal production and/or plasma concentration of

1,25(OH)2D3 has also aroused controversy. Some findings (Abe et al. 1979; Nys et al. 1986a; Nys et al. 1992c) indicated that renal production or plasma concentration increased 10 to 20 h post oviposition, in accordance with the diminished plasma Ca2+ and increased plasma PTH levels (van de Velde et al. 1984; Nys et al. 1986a; Singh et al. 1986; Frost et al. 1990; Ieda et al. 2000; Sugiyama et al. 2001; Goto et al. 2002b). However, other studies did not support these findings (Fig. 3; (Montecuccoli et al. 1977b; Sedrani et al. 1977; Bar et al. 1984b;

Kaetzel et al. 1985; Soares et al. 1988)), or even suggested that 1,25(OH)2D3 production decreased during shell calcification (Kenny 1976). This controversy could arise from the duration of the ESG inactivity period during the “pause” day at the end of the clutch

(Montecuccoli et al. 1977b; Sedrani et al. 1977). Nevertheless, even if 1,25(OH)2D3 synthesis is really stimulated during shell formation, this could induce either a non-genomic or a delayed rather than an acute genomic response, because the genomic response takes several hours to become evident (Lawson 1978). 14

Fig. 3. Vitamin D metabolism and calbindin expression during the egg cycle.  , calbindin mRNA;  , calbindin; , shell calcium; , calcium absorption capability; , normal cycle; , day of pause at the end of the clutch (no shell formation). Mean ± SE. Means designated by different letter are significantly different (P < 0.05) (with permission of: (Bar et al. 1976a; Montecuccoli et al. 1977b; Bar et al. 1992a; Bar et al. 1992d)).

The comparison and interpretation of experimental results obtained from laying birds is quite difficult and may lead to mistaken or controversial interpretations because, whereas growth is a continuous process, egg formation and shell calcification are circadian in nature. This can account for some of the aforementioned disagreements in the findings concerning vitamin D metabolism and expression in the laying hen. Since the Ca2+ demand is the major

2+ factor regulating 1,25(OH)2D3 synthesis, any change in Ca demand will affect 1,25(OH)2D3 synthesis. The loss of a massive proportion of the body Ca2+ in the laid eggshell induces the development of a physiological Ca2+ deficiency and acceleration of vitamin D metabolism. The overall loss of Ca2+ depends on the rate of egg production and on the Ca2+ content of each eggshell, the latter characteristic being independent of the laying rate. Both characteristics are easily affected by nutrition, environment and disease. 15

4. Vitamin D expression Vitamin D is expressed in all four tissues associated with calcium homeostasis in the laying bird. However, the present review will focus on the expression of the major transporting tissues, the intestine and the ESG, rather than on the kidney or bone (reviewed in (Hurwitz 1992; Norman et al. 1993; Gay 1996; Edwards 2000; Gay et al. 2000; Sugiyama et al. 2001; Whitehead 2004)).

2+ The action of 1,25(OH)2D3 on the Ca -transporting cells is initiated by its binding to vitamin D receptors (VDRs), which have been identified in the mammalian and the avian intestine and kidney (reviewed in (Pike 1997; Haussler et al. 1998; Holick 2003; DeLuca 2004; Young et al. 2004; Dusso et al. 2005; Norman 2006)), and in the avian ESG (Coty 1980; Takahashi et al. 1980), and the mammalian uterus and placenta, bone, parathyroid glands (PT), pancreas, cardiovascular system and in other tissues (reviewed in (Pike 1997; Walters 1997; Haussler et al. 1998; Dusso et al. 2005)).

Free 1,25(OH)2D3 is transferred into the nucleus of the target cell, where it binds to the

VDR. The binding of 1,25(OH)2D3 to VDR stimulates the synthesis of RNAs coding for several proteins. Among these proteins are the vitamin-dependent calcium-binding proteins, calbindin D28K (Wasserman et al. 1966; Bar et al. 1972a) or calbindin D9K (Bronner et al. 1975; Thomasset et al. 1981) in the avian or mammalian intestine, respectively; Ca2+ATPase (calcium pump, PMCA) (reviewed in (Stokes et al. 2003; Strehler et al. 2007)); and epithelial calcium channels (TRPVs) proteins (reviewed in: (Belkacemi et al. 2005; Hoenderop et al. 2005b; Niemeyer 2005)). These three groups of proteins appear to facilitate the transcellular mechanism of Ca2+ transport (reviewed in ((Bouillon et al. 2003; Bronner 2003; Wasserman 2004; Hoenderop et al. 2005b)). Many other genes/proteins appear also to be vitamin D dependent or related (vitamin D-induced/suppressed genes or those having VDREs). Among them are osteocalcin and osteopontin, collagen type I (reviewed in (Kream et al. 1997; Pike 1997; Haussler et al. 1998)), and carbonic anhydrase II (CA; (Drewe et al. 1988; Quelo et al. 1994). Among the latter, only CA (believed to be associated with Ca2+ transport in the ESG) will be addressed in this review.

The formation of 1,25(OH)2D3 and its interaction with the nucleus are relatively slow. Whereas the response to the other key calcium-regulating hormone, PTH, is rapid (within minutes), the genomic effect of vitamin D or of its active metabolites requires 9 to 24, or 3 to 6 h, respectively (reviewed in (Lawson 1978)). 16

In addition to the slow genomic effects of the active metabolites of vitamin D, a rapid effect of 1,25(OH)2D3 on a vesicular transport mechanism that involves microtubules and microtubule-associated calbindin (transcaltachia) was reported by (Nemere et al. 1984), and it was extensively studied since then (reviewed in (Nemere et al. 1990; Norman et al. 2002; Larsson et al. 2003; Dusso et al. 2005)). Such a rapid response (within seconds to minutes) suggested that non-genomic responses were involved. Additional non-genomic responses were demonstrated for brush border membrane permeability, composition or fluidity (Bikle et al. 1980; Putkey et al. 1982; Rasmussen et al. 1982; Brasitus et al. 1986) (see also 5.1). The overall contribution of the rapid non-genomic responses to intestinal or ESG Ca2+ transport is not yet known. Among the proteins considered to be vitamin D dependent, only calbindin has been widely studied in birds; VDR and the other vitamin-dependent proteins have been less studied in birds in general, and much less studied in laying birds. Furthermore, most of the relevant studies on vitamin D derivatives in laying birds addressed their involvement in production traits rather the biological mechanisms associated with their activity.

5. Vitamin D-dependent proteins 5.1. Vitamin D receptor

The binding of 1,25(OH)2D3 to VDR activates the formation of a heterodimer of VDR with a partner protein such as retinoid X receptor (RXR). Following the binding of

1,25(OH)2D3 to a VDR, the receptor is phosphorylated, and recruits an unaligned retinoic X receptor (RXR) to form a VDR-RXR heterodimer. The RXR-VDR-1,25(OH)2D3 complex recognizes and targets the gene through a high-affinity association with the VDREs in the gene-promoter region, and thereby stimulates the synthesis of RNAs coding for several proteins. VDREs were identified in the promoters of gene-coding calbindins, 24-hydroxylase, osteopontin, osteocalcin and others (reviewed in (DeLuca 2004)), and in PMCAs, (Glendenning et al. 2000), TRPVs (Weber et al. 2001) and CA (Quelo et al. 1994). However, some of the available data are not fully consistent with the hypothesis that all the isomers of PMCAs, TRPVs and calbindins are vitamin D dependent in all examined species or tissues. In the chicken, the intestinal VDR content is markedly increased during maturation, prior to the onset of egg production. At the onset of egg production it remains high or declines slightly, but still remains higher than in the immature female chick (Wu et al. 1994). Maturation or estradiol treatment induce the development of immature ESG tissues and markedly increase their VDR content (Striem et al. 1989; Striem 1990; Bar et al. 1992c; 17

Yoshimura et al. 1997). The ESG VDR content remains high also after the onset of production. This high content is maintained during a short (up to 9 d) chemically induced egg arrest (Bar et al. 1990b) or during a longer arrest caused by molt induction, although the mucosal tissue is regressed (Yoshimura et al. 1997). In the normal laying hen the ESG VDR concentration is 33 to 20% of the intestinal VDR content (Coty 1980; Bar et al. 1984b) and its gene expression oscillates during the diurnal egg cycle in close temporal association with eggshell calcification (Ieda et al. 1995). In addition to the nuclear VDR, membrane-bound receptor(s) (mVDR) linked to signal transduction appear to mediate the non-genomic rapid responses to 1,25(OH)2D3 and

24,25(OH)2D3 (reviewed in (Norman et al. 2002; Dusso et al. 2005; Norman 2006); see also 3.1 & 4). The nature of these receptors remains controversial. At least two distinct proteins have been identified: the thiol-dependent oxidoreductase ERp57 (1,25D3-MARRS), and annexin II. However, the involvement of the latter is not fully accepted. On the other hand, it was recently shown that in some cells the nuclear VDR is associated with caveolae that are present in the plasma membrane. Recently (Norman 2006) proposed a conformational ensemble model to describe how changes in ligand shapes of 125(OH)2D3 act through the VDR in different cellular locations, and can selectively mediate both genomic and non- genomic rapid responses of the VDR.

5.2. Calbindins Expression of this group of proteins, previously named CaBPs (Ca-binding proteins) differs (Table 1) among many cell types and species. High concentrations of calbindins are found in tissues characterized by their massive transport of Ca2+, such as intestine, kidney, placenta, uterus and ESG of birds (Wasserman et al. 1966; Taylor et al. 1967; Corradino et al. 1968; Bruns et al. 1978; Marche et al. 1978; Delorme et al. 1983). Lower concentrations of calbindins are found in other tissues associated with Ca2+ homeostasis and metabolism, such as bone, tooth cells and PT cells. These proteins are found also in tissues not directly associated with Ca2+ transport: the nervous tissues contain high concentrations, and the pancreas and testes contain low concentrations (reviewed in (Christakos et al. 1997; Thomasset 1997)). Whereas the mammalian intestine, uterus, placenta, and other tissues

(including the kidney in a few species) contain mainly calbindin D9K, the avian and other lower species' tissues contain calbindin D28K. In the chick intestine and ESG, the protein is localized primarily in the absorptive cells and in the tubular gland cells, respectively (Lippiello et al. 1975; Jande et al. 1981; Wasserman et al. 1991). In avian species high 18

concentrations of calbindin was found in the intestine, kidney and the ESG (Wasserman et al. 1966; Taylor et al. 1967; Corradino et al. 1968). In the chicken and quail intestine, calbindin concentrations are higher in the proximal than in the distal segments (Taylor et al. 1967; Bar et al. 1976b). The calbindins are considered to facilitate the movement of Ca2+ inside the epithelial cells of the calcium-transporting organs. Although these proteins appear to be

2+ primarily associated with Ca transport, they may also be involved in protecting the cells from high concentrations of Ca2+ or from cellular degradation via apoptosis (Christakos et al. 2003). In all three major Ca2+-transporting organs, the intestine, the kidney and the ESG of birds, concentrations of calbindin is closely correlated with Ca2+ transport (Taylor et al. 1969; Morrissey et al. 1971; Bar et al. 1975a; Bar et al. 1979a; Bar et al. 1984b). However, whereas the calbindin contents in the kidney and in the ESG are correlated with the mass of calcium transported (weight unit), the intestinal calbindin is correlated with calcium transport capability (% of absorption) (Fig. 43; (Bar et al. 1979b)).

Fig. 4. Plasma calcium, intestinal and plasma calbindin, and intestinal calcium absorption as functions of calcium intake (from (Bar et al., 1979b); CaBP, calbindin; Ca, total calcium).

3 In the final proof figs 4 & 5 were switched over. 19

5.2.1. Calbindin and vitamin D: The calbindin gene promoter contains a VDRE (reviewed in (Christakos et al. 1997; DeLuca 2004))4. Three species of mRNAs are encoded in the avian intestine, kidney and ESG by the calbindn D28K gene. A major species (approximately 2.0 kb) and two minor ones (approximately 2.7 and 3.0 kb (Fig. 5c; lanes 1 & 2). All three species of mRNA are induced by 1,25(OH)2D3 in the avian intestine and kidney, but not in the ESG (Hunziker 1986; Mayel-Afshar et al. 1988; Bar et al. 1990a; Striem et al. 1991; Bar et al.

1992a). Intestinal calbindin synthesis reflects the changes in 1,25(OH)2D3 in the intestinal cell. These include the changes that result from exogenous supplementation of vitamin D or its derivatives (Wasserman et al. 1966; Bar et al. 1975b; Bar et al. 1976b; Bar et al. 1978b; Bar et al. 1990b; Striem et al. 1991; Bar et al. 1992a), dietary restriction of Ca2+ and/or P in non-laying birds (Wasserman et al. 1968; Morrissey et al. 1971; Bar et al. 1972b; Friedlander et al. 1977; Montecuccoli et al. 1977a), growth and age (Bar et al. 1981a; Bar et al. 2003). As in the intestine, the full modulation of renal calbindin mRNAs requires vitamin D metabolites, without which renal calbindin mRNAs levels are very low and are almost unaffected by dietary alterations. However, unlike intestinal calbindin, the renal calbindin, although significantly diminished in vitamin D-deficient chicks, did not completely disappear, and even retained part of its capability to be modulated in response to dietary alteration (Bar et al. 1975b; Bar et al. 1990a).

Most of the available evidence does not support the idea that ESG calbindin D28k is vitamin D dependent: endogenous or exogenous 1,25(OH)2D3 (Bar et al. 1976b; Bar et al. 1988; Bar et al. 1990b) had no effect on ESG calbindin, whereas it did affect intestinal or renal calbindin; and ESG calbindin mRNAs was induced in the shell-forming, vitamin D- deficient quail that was fed high dietary Ca2+ (Striem et al. 1991). Furthermore, dietary calcium that affected intestinal and renal calbindin, or P restrictions that affected intestinal and renal calbindin in accordance with its effects on vitamin D metabolism, did not affect synthesis of ESG calbindin or calbindin mRNAs (Bar et al. 1973b; Bar et al. 1978b; Bar et al. 1984a; Bar et al. 1984b; Bar et al. 1999; Ieda et al. 1999). Similarly, maturation prior to the onset of laying, associated with increased renal formation of 1,25(OH)2D3, induced the synthesis only of intestinal calbindin and calbindin mRNAs, but not of ESG or renal calbindin (Bar et al. 1972a; Bar et al. 1973b; Bar et al. 1978b; Bar et al. 1990b; Bar et al. 1992a; Bar et al. 1999; Goto et al. 2002b).

4 This sentence was removed from the final proof. 20

On the other hand, the presence of a considerable concentration of VDR in the ESG (Coty 1980; Bar et al. 1984), as well as the parallel fluctuations of ESG VDR (Ieda et al. 1995) and calbindin mRNAs during the egg cycle, are indicative of possible involvement of

1,25(OH)2D3 in ESG functionality (Bar et al. 1984b; Bar et al. 1990b). This idea is further supported by the single finding that injection of 1,25(OH)2D3 directly into the ESG lumen increased the ESG calbindin concentration (Ohira et al. 1998), and is also supported by the finding that 1,25(OH)2D3 has a slight effect on synthesis of ESG calbindin mRNA in vitro, or of calbindin in vivo in estrogen-treated immature female chicks (Corradino 1993; Corradino et al. 1993).

Fig. 5. Visualization of calbindin RNAs from the duodenum and eggshell gland (ESG) by Northern blotting. (a) Effect of laying on duodenal calbindin RNAs: Lane 1, vitamin-D-deficient chick; lane 2, mature non-laying; lane 3, laying hen (from (Bar et al. 1992a)). (b) Effect of laying on ESG calbindin RNAs: Lane 1, laying hen duodenum; lane 2, ESG of mature non-laying; lane 3, ESG of laying hen during period of ESG inactivity; lane 4, ESG of laying hen during period of shell calcification (from (Bar et al. 1992a)). (c) Effect of shell calcification blocking of ESG calbindin RNAs: Lane 1, untreated laying hen; lane 2, laying hen treated with a single oral dose of the carbonic anhydrase inhibitor acetazolamide; lane 3, laying hen following forced immature oviposition (from (Bar et al. 1992b)). (d) Effect of shell quality: Lane 2, laying hen with shell-less eggs; lane 3, laying hen with normal eggs; lane 4, laying hen with broken or cracked eggs; above are shown the previous last eggs. Hens were sampled 17 h post oviposition (from (Bar et al. 1992b). Membranes were hybridized with oligonucleotide complementary to the mRNA sequence encoding amino acids 58-68 of chicken calbindin (with permission). Similar results were obtained using the quantitative “solution hybridization assay". 21

5.2.2 Reproduction and gonadal hormones: In the female bird intestinal calbindin mRNA (Striem et al. 1991; Bar et al. 1992a) and calbindin (Bar et al. 1972a; Montecuccoli et al. 1977b; Bar et al. 1978a; Bar et al. 1981b; Bar et al. 1990b; Bar et al. 1992a; Nys et al. 1992a; Wu et al. 1994; Bar et al. 1996) are moderately increased during sexual maturation. Onset of laying markedly increased calbindin mRNA (Nys et al. 1989; Bar et al. 1990b; Striem et al. 1991; Bar et al. 1992a; Nys et al. 1992a) and calbindin (Wasserman et al. 1968; Bar et al. 1973b; Bar et al. 1976a; Bar et al. 1978a; Bar et al. 1978b; Bar et al. 1992a) (Sugiyama et al. 2007) synthesis in the intestine and ESG. Molting (Yoshimura et al. 1997; Yosefi et al. 2003) and any other factor that arrests egg production (Bar et al. 1973b; Bar et al. 1973a; Bar et al. 1992a) markedly reduce the intestinal and ESG calbindin contents.

A combined treatment of estradiol and testosterone may mimic the effect of maturation on intestinal calbindin (Bar et al. 1979a; Nys et al. 1984c; Striem et al. 1989; Striem 1990; Nys et al. 1992a) in vitamin D-fed female chicks, most likely indirectly, as a result of the increased demand for calcium (see 2.2) The role of gonadal hormones in the development of the oviduct is well established, and this raised an interest in their role in synthesis of ESG calbindin and other proteins. Whereas uterine calbindin D9K in mammals, but not calbindin D28K in mice (Gill et al. 1995), is noticeably induced by estrogens (reviewed in (Choi et al. 2005)), most researchers (Navickis et al. 1979b; Nys et al. 1989; Striem et al. 1989; Bar et al. 1990b; Striem 1990; Nys et al. 1992b; Corradino 1993; Corradino et al. 1993; Bar et al. 1996), but not all of them (Goto et al. 2002a), suggest that calbindin D28K synthesis in the avian ESG is only slightly induced by estrogens or sexual maturation. The observed maximal increment in ESG calbindin or its mRNAs, in response to maturation or estrogen treatment is one to two orders of magnitude smaller than that induced by shell calcification in the normal laying bird. These findings suggest that estrogens alone cannot account for the markedly elevated synthesis of calbindin mRNAs in the ESG of the shell-calcifying bird. On the other hand, the repetitive small effects of estrogens, together with the occurrence of an estrogen-responsive element on the 5'- flanking region of the calbindin D28K promoter (Gill et al. 1995), support the idea that estrogens are involved in the mechanism of calbindin gene expression in the ESG. In addition, indirect effects may result from the influence of estrogens on oviduct development during maturation or from estradiol treatments (Striem 1990; Corradino et al. 1993; Berg et al. 2001). 22

Whereas testosterone does not affect ESG calbindin synthesis, progesterone and dexamethasone inhibit it (Bar et al. 1996; Goto et al. 2002a). Dexamethasone, but not progesterone, also inhibited synthesis of intestinal calbindin and its mRNA. Both prolonged the egg cycle but, whereas dexamethasone increased shell calcification, progesterone inhibited Ca2+ deposition onto the shell. This suggests that the effect of progesterone is specific and may be involved in the regulation of ESG calbindin synthesis. This hypothesis is supported by the finding that the plasma progesterone concentration oscillated during the diurnal egg cycle (Doi et al. 1980; Johnson et al. 1980; Nys et al. 1986a; Braw-Tal et al. 2004), in close temporal association with eggshell calcification, and peaked about 4 h before the next ovulation, at the time when shell calcification diminished and then stopped. The ESG remains in a refractory state prior to actual reproduction, and calbindin mRNAs (Nys et al. 1989; Striem et al. 1991; Bar et al. 1992a; Ieda et al. 1995) and calbindin (Bar et al. 1973b; Bar et al. 1978a; Bar et al. 1978b) begin to appear during calcification of the first eggshell. In the ESG, calbindin mRNAs oscillate during the diurnal egg cycle, between near zero and high concentrations, in close temporal association with eggshell calcification (Figs. 3, 5). Similarly to duodenal-calbindin, ESG-calbindin is lower in non-laying hens during molting (Yoshimura et al. 1997; Yosefi et al. 2003) or in hens that lay shell-less eggs (Nys et al. 1986b; Rabon et al. 1991; Bar et al. 1999) than in those that lay calcified eggs, and is lower in hens that form thin eggshells than in those that form thick ones (Bar et al. 1984b; Rabon et al. 1991; Bar et al. 1992b; Goto et al. 2002b). This suggests that Ca2+ transport in the ESG, similarly to that in the kidney, plays a major role in the synthesis of ESG calbindin mRNAs. The absence of a major effect of vitamin D or estrogens on ESG calbindin, the association of ESG calbindin mRNAs, but not of calbindin, with shell formation, and the relationship of ESG calbindin with the mass of Ca2+ transported, rather than with the transport capability (as in the intestine), suggests that Ca2+ transport in the ESG, similarly to that in the kidney, plays a major role in the synthesis of ESG calbindin mRNAs. In light of the above findings, it was hypothesized that the regulative mechanism for the synthesis of calbindin mRNAs in the ESG is a complex, multiple one, which involves, in addition to a major Ca2+-transport-related process, estrogen and other endocrine factors (Nys et al. 1989; Striem et al. 1991; Bar et al. 1992a; Nys et al. 1992b; Corradino 1993; Corradino et al. 1993; Bar et al. 1996; Bar et al. 1999). 23

5.3. Plasma Membrane Calcium-ATPase (Ca2+ATPase Or PMCA) The PMCAs use the energy stored in ATP to extrude Ca2+ out of the cell against the electrochemical gradient. This group of more than 30 isomers is encoded by at least four independent genes (PMCA1-4) (reviewed in: (Carafoli 1992; Howard et al. 1993; Bouillon et al. 2003; Belkacemi et al. 2005; Hoenderop et al. 2005b; Nijenhuis et al. 2005)) with two alternative, independent splicing sites. Whereas PMCA1 and PMCA4 are widespread, PMCA2 and PMCA3 are tissue specific. The PMCA1b is the predominant isomer expressed in the mammalian (reviewed in: (Howard et al. 1993; Nijenhuis et al. 2005)) and the chicken intestine (Melancon et al. 1970; Strittmatter 1972; Davis et al. 1987) and kidney (Qin et al. 1993a). In the intestine, kidney and placenta the PMCAs are located on the basolateral membrane of the epithelial cell toward which Ca2+ is transported (Borke et al. 1989a; Borke et al. 1989b; Borke et al. 1990). The specific involvement of the intestinal Ca2+ pump and PMCA genes in intestinal Ca2+ transport has been intensively studied, mostly in mammals (reviewed by (Hoenderop et al. 2002; Stokes et al. 2003; Hoenderop et al. 2005b)) and in non-laying birds (Davis et al. 1987; Wasserman et al. 1992; Cai et al. 1993; Pannabecker et al. 1995). The intestinal expression at the transcriptional level is modulated by vitamin D (reviewed in: (Zelinski et al. 1991; Wasserman et al. 1992), and also by a variety of factors that affect vitamin D metabolism (see 3.2), such as alterations in dietary Ca2+ and P concentrations (Wasserman et al. 1992; Cai et al. 1993; Armbrecht et al. 1994), age (Armbrecht et al. 1994), pregnancy and lactation in mammals (Zhu et al. 1998). Recently a VDRE sequence was identified in the PMCA1 gene (Glendenning et al. 2000). All these findings support the idea of vitamin-D-mediated modulation of the intestinal PMCA. On the other hand, a consistent stimulatory effect of

1,25(OH)2D3 on renal PMCA1 has not been established: 1,25(OH)2D3 did not stimulate, or even down-regulate renal PMCA1 (reviewed by (Hoenderop et al. 2005b)). There is little evidence to support the idea that estrogens also regulate PMCAs: In estrogen receptor knockout mice, renal, but not intestinal, PMCA was significantly reduced (Van Cromphaut et al. 2003). In ovariectomized mice, 17-estradiol supplementation induced intestinal PMCA1b mRNA expression, but to a lesser degree than TRPV(5 and 6), calbindin (Van-Abel et al. 2003), and PMCA activity in kidney membrane vesicles (Dick et al. 2003). Although less information is available with regard to the role of PMCAs in intestinal absorption of Ca2+ in laying birds, it is most likely similar to those in mammals and non- laying birds. At least one study (Grunder et al. 1990) indicated that vitamin D 24

supplementation slightly increased the intestinal (jejunal) Ca2+ATPase activity in laying hens. Immunohistochemical and enzymatic evidence indicate that PMCAs are also present in the ESG of laying birds (Pike et al. 1975; Coty et al. 1982; Lundholm 1982; Grunder et al. 1990; Wasserman et al. 1991). Early evidence suggested that energy-dependent Ca2+ transport took place across the ESG wall (Schraer et al. 1970), where the PMCA is localized primarily in the apical-microvillar membrane of the tubular gland cells (Yamamoto et al. 1985; Wasserman et al. 1991; Arai et al. 1996) facing the ESG lumen, where shell calcification occurs. Unlike intestinal or renal PMCA, ESG PMCA was not modulated by 1,25-(OH)2D3 (Nys et al.

1984a) or was only slightly affected by it (Grunder et al. 1990). Estradiol treatment (Corradino et al. 1993; Qin et al. 1993b) stimulated ESG, but not intestinal (Qin et al. 1993a) ATPase activity or concentration, independently of the vitamin D status (Nys et al. 1984a; Corradino et al. 1993). Egg laying increased (Pike et al. 1975) and suppression of shell formation decreased the activity of PMCAs in the ESG. The association of PMCAs with the egg cycle remains controversial (Grunder 1983; Watanabe et al. 1989; Balnave et al. 1992). Thinning of the eggshell caused by p-p'-DDT and DDE in several species of birds was associated with a reduction in the ESG Ca2+ATPase activity. The accumulated data on the association between Ca2+ATPase and the loss of eggshell integrity in birds exposed to environmental pollution has raised special interest in the environmental aspects of this association (reviewed in (Lundholm 1997), see also 5.5).

5.4. Epithelial calcium channels (TRPVs) The TRP (Transient Receptor Potential) super-family of cations channel is widely distributed in animal tissues; they comprise more than 27 proteins involved in the transport of ions into cells in mammals. Their six subfamilies include the TRPVs (reviewed in: (Belkacemi et al. 2005; Hoenderop et al. 2005b; Niemeyer 2005; van de Graaf et al. 2007; Venkatachalam et al. 2007)). Two apical Ca2+ channels – TRPV5 and TRPV6, also known as

ECaC1 and ECaC2 or CaT2 and CaT1, respectively – were identified in the 1,25(OH) 2D3- responding epithelia of the upper intestine, the distal nephron, bone, and the mammalian placenta and uterus (reviewed in (Peng et al. 1999; Belkacemi et al. 2005; Hoenderop et al. 2005b; van der Eerden et al. 2005; Lambers et al. 2006), all of which are characterized by massive Ca2+ transport. Studies with VDR knockout mice suggested that expression of the

2+ Ca channel genes in the intestine and kidney are regulated by 1,25-(OH)2D3, and that the genes encoding them have a VDRE (reviewed in (Belkacemi et al. 2005; Brown et al. 2005; Hoenderop et al. 2005b)). Although most findings support the idea that TRPVs are 25

1,25(OH)2D3 dependent, another study (Weber et al. 2001) opposed this idea. Estrogens have a distinct, vitamin D-independent stimulating effect at the genomic level of TRPV6 (reviewed in (Hoenderop et al. 2005a; van Abel et al. 2005)); they also affect renal TRPV5 (Van Cromphaut et al. 2003). The effect of estrogen on TRPV6 may be mediated by an estrogen-responsive element (ERE) that has been identified on the promoter sequence of mouse TRPV6, but not on TRPV5 (Weber et al. 2001). However, the same study also suggested that the effects of estrogen on intestinal calcium absorption and renal calcium reabsorption are not mediated through altered TRPV expression, at least at the transcriptional level. Recent evidence is also indicative of estrogen dependency of TRPV6 expression in the mouse uterus (Lee et al. 2007). TRPV5 and TRPV6 are considered to facilitate the entry of Ca2+ into the epithelial cells of the calcium-transporting organs. The specific role of TRPVs in the transport of Ca2+ in the laying-hen intestine and ESG has not yet been studied.

5.5. Carbonic anhydrase The carbonic anhydrases (CA) are a group of zinc-containing enzymes that catalyze the reversible hydration of carbon dioxide. The CAs are involved in bone resorption and calcification, ion transport, acid-base metabolism, and the movement of respiratory gases. The CA family comprises three evolutionarily unrelated subfamilies without significant sequence homology. At least 16 different isomers were identified in mammalians (reviewed in (Esbaugh et al. 2006; Purkerson et al. 2007)) and several novel isozymes have also been identified in avians (Holmes 1977). The commonest of these isomers in avian tissues is CA-II (Holmes 1977). CAs were found in the avian kidney (Holmes 1977; Brown et al. 1982; Gabrielli et al. 1998), epiphysis (Dulce et al. 1960) – specifically in bone osteoclasts (Gay et al. 1974; Billecocq et al. 1990) – intestine (Nys et al. 1984b; Grunder et al. 1990; Gabriella et al. 1994) and ESG (Benesch et al. 1944; Bernstein et al. 1968) – specifically in the ESG epithelial cells (Arai et al. 1996). The CAs are present in other cells also, such as gastric

+ - parietal cells and salivary glands, where their main role is to generate H and HCO3 during

- acid-base regulation. The formation of HCO3 in the avian ESG appears to be of especial importance for the formation of CaCO3 in the shell, where it acts as the sole counter-ion for Ca2+. The dependency of CA on vitamin D is not yet clear: a VDRE region was identified in the CA-II gene (Quelo et al. 1994), and 1,25(OH)2D3 was found to regulate the transcription of CA-II in myelomonocytes (Lomri et al. 1992), and to induce their differentiation

((Billecocq et al. 1990; Lomri et al. 1992). Vitamin D3 deficiency caused a reversible 26

reduction in CA activity in the ESG of the laying hen (Grunder et al. 1990), but CA activity was found to be unrelated to plasma level, or to exogenous supplementation of 1,25(OH)2D3 (Nys et al. 1986b; Grunder et al. 1990). CA inhibitors, such as acetazolamide, rapidly (within 10 to 12 h post administration) blocked shell calcification (Bernstein et al. 1968; Pearson et al. 1977; Eastin et al. 1978; Bar et al. 1992a; Bar et al. 1999) in spite of the availability of dietary calcium and Vitamin D3, and reduced ESG and intestinal calbindin (Bar et al. 1992a; Bar et al. 1999) and ESG Ca2+- ATPase (Lundholm 1990). Of interest is the fact that acetazolamide in vitro reduced CA activity, but not Ca2+ transport (Ehrenspeck et al. 1971). This is indicative of an indirect role of CA in Ca2+ transport. Thinning of the eggshell caused by p-p'-DDT and DDE in quail was associated with a reduction in the ESG CA activity (Bitman et al. 1970).

6. Vitamin D-controlled transcellular transport of Ca2+ In birds and mammals the overall Ca2+ transport reflects a sum of saturated and unsaturated processes. Whereas the saturated process corresponds to active transcellular transport, the unsaturated process consists of diffusion through a paracellular route (reviewed in (Wasserman 1997; Bouillon et al. 2003; Bronner 2003; Wasserman 2004; Hoenderop et al. 2005b; van de Graaf et al. 2007)). Briefly, the transcellular transport consists of three major steps: entry of Ca2+ through the brush border, diffusion or movement to the basal membrane, and extrusion through the basal membrane. The first step proceeds down the chemical gradient of Ca2+ that results from its low cellular concentration (10-7 M) and high plasma and intestinal lumen concentrations (>10- 3 M). This step appears to be facilitated by TRPV6 (see 5.4) and to a lesser extent by TRPV5. The second step appears to be facilitated by intracellular calbindins (Feher 1984; Feher et al. 1992; Koster et al. 1995). There is some evidence for calbindin-mediated cytosolic-free diffusion of Ca2+ and of vesicular transport (Nemere 1992).. The energy-dependent third step proceeds up the chemical gradient of Ca2+ that results from the low cellular Ca2+ concentration and the higher plasma Ca2+ concentration (1.25 to 1.50 mM). This step is facilitated by PMCA (see 5.3) and, to a lesser extent, by a sodium-calcium (Na+/Ca2+) exchanger (reviewed in (Hoenderop et al. 2005b; Lytton 2007)). Components of all three steps of transcellular transport are vitamin D-dependent (see 5), because TRPV6 and TRPV5, calbindins and PMCA all comprise a VDRE and/or are unaffected in the tissues of VDR- knockout animals, and/or are stimulated in normal animals by 1,25(OH)2D3 or by factors that 27

affect its formation, such as dietary Ca2+, age, pregnancy and lactation in mammals, or egg laying in birds. Doubt is cast on the significance of transcellular transport in the intestine of the laying hen by the high Ca2+content of the intestinal lumen, because the electrochemical potential difference (ECPD) gradients in laying birds fed commercial high-calcium diets is sufficient to ensure a massive paracellular passive transport (Hurwitz et al. 1968) through the tight junctions. The possible involvement of 1,25(OH)2D3 in paracellular transport through the tight junctions is still a matter of controversy (McCormick 2002; Wasserman 2004). This issue is to be addressed and discussed with regards to the laying birds in another manuscript (Bar 2009). The findings of recent studies indicate that it is possible to control the tight junction (TJ) proteins (that connect between epithelial cells and form a biological barrier), permeability and paracellular transport (reviewed in (Schneeberger et al. 2004; Van Itallie et al. 2006)). It

2+ appears that 1,25(OH)2D3 and VDR, as well as cellular Ca , are among the factors responsible for preserving the integrity of TJ complexes, up-regulating TJ proteins, and modulating TJ permeability and paracellular Ca2+ transport (Tang et al. 2003; Kutuzova et al. 2004; Fujita et al. 2008; Kong et al. 2008). These findings support the hypothesis that

2+ 1,25(OH)2D3, also, may regulate paracellular Ca transport. In the ESG, although TRPVs were not yet identified, the presence of the other two components (PMCA and calbindin) of transcellular transport Ca2+, the localization of PMCA (see 5.3), the presence of TRPVs in mammals uterus and placenta (reviewed in (Lee et al. 2007)) and the apparently uphill transport of Ca2+ that occurs there, support the idea that Ca2+ is transported via the transcellular pathway. However, the lack of an effect of vitamin D on the ESG transport of Ca2+, and the obvious and critical involvement of CA in this procedure, suggest that the mechanisms there could not be easily explained solely in terms of simple trancellular and/or paracellular mechanisms of Ca2+ transport. This issue is to be addressed and discussed extensively in another manuscript (Bar 2009).

6. Concluding remarks Shell calcification of birds with long clutches imposes severe demands on Ca2+ homeostasis, which is maintained by the highly efficient Ca2+ transport mechanisms that operate in the intestine, bones and the ESG. As an external Ca2+ supply is not guaranteed throughout shell-calcification period, these birds extract a significant portion of the shell Ca2+ 28

from the medullary bone reserves. Therefore, within a period of one egg cycle, the intestine not only supplies the major proportion of the shell Ca2+; it also replenishes most of the Ca2+ removed previously from the bone. Vitamin D is the major factor regulating intestinal transport. Its metabolism in laying birds is similar to that in non-laying higher vertebrates, although the birds do not use vitamin

D2 metabolites efficiently, and their metabolism is somewhat differently regulated by plasma P. The birds' Ca2+ needs, as manifested through their plasma and PTH Ca2+ contents, appear to form the most important regulatory factor of vitamin D metabolism. Therefore, in these birds any factor capable of modifying Ca2+ demands, such as the clutch length, the shell mass and the egg cycle, consequently modulates vitamin D metabolism. This leads to some disagreements regarding interpretation of the findings on vitamin D metabolism obtained in different studies. Vitamin D expression in the intestine (the bone is not reviewed here) appears also to be similar to that known in non-laying higher vertebrates. Although the TRPVs have not yet been identified in birds, it most likely that the transcellular Ca2+ transport is also similar. However, because of the high calcium intakes of these birds, the paracellular pathway appears to be more important, especially during the period of shell calcification. The recent findings on the effect of calcium and vitamin D metabolites on TJ proteins and permeability offer new insights into Ca2+ transport in laying birds. The importance of vitamin D for ESG Ca2+ transport is less well understood and more complicated (ESG transport will be discussed in a later review; in preparation): although many of the vitamin D-dependent proteins are present in the ESG, in most cases, they do not respond to vitamin D. Some of them, such as the vitamin D-dependent calbindin, are differently regulated, and not necessarily in a vitamin D-dependent manner. On the other hand, ESG CA, which is most likely also a vitamin D-dependent protein, may play a determinant role in this tissue.

Acknowledgments Contribution from the Institute of Animal Science, Agricultural Research Organization, the Volcani Center, Bet Dagan, Israel: No. 516-08. Encouragement and comments by Prof. S. Yahav, ARO, are acknowledged with appreciation. 29

References