Parathyroid Hormone-Related Protein: A Regulated Calcium-Mobilizing Product of the Mammary Gland

Parathyroid Hormone-Related Protein: A Regulated Calcium-Mobilizing Product of the Mammary Gland MARK A. THIEDE Pfizer Central Research Groton, CT 06340 ABSTRACT

Parathyroid hormone-related protein shares similarities in sequence and function with the endocrine hormone, parathyroid hormone. However, unlike parathyroid hormone, a product of the parathyroid glands, parathyroid hormone-related protein has a wide distribution in tissues, including the mammary gland. Although during pregnancy the expression of parathyroid hormonerelated protein in the mammary gland is low, following birth, protein levels rise sharply in the gland in response to elevations in serum prolactin. Large amounts of parathyroid hormone-related protein are secreted into milk, suggesting a possible role in the neonate. Transient phosphaturia and elevations of parathyroid hormone-related protein in mammary vein plasma support a possible endocrine function for parathyroid hormone-related protein during lactation. Recent evidence suggests a local function for parathyroid hormone-related protein in the lactating mammary gland, and evidence exists that parathyroid hormone-related protein stimulates calcium secretion by the goat mammary gland. Parathyroid hormonerelated protein, a putative vasodilator, is produced by the external nutrient vasculature of the mammary gland, and levels within this tissue are regulated during lactation. Infusion of parathyroid hormone-related protein into the ovine mammary artery increases gland blood flow, suggesting a role for the protein in modulation of mammary gland hemodynamics. Regulation of parathyroid hormone-related protein synthesis by the lactating gland, together with

Received June 16, 1993. Accepted October 18, 1993. 1994 J Dairy Sci 77:1952-1963

the protein's actions on regional blood flow and calcium secretion, support an important function in the mammary gland during lactogenesis. (Key words: parathyroid hormonerelated protein, mammary gland, calcium secretion, lactation)

Abbreviation key: PTH = parathyroid hormone, PTHrP = parathyroid hormone-related protein INTRODUCTION

In 1941, Fuller Albright (2) at the Massachusetts General Hospital postulated that the hypercalcemia associated with certain cancers resulted from the tumoral secretion of a substance similar to parathyroid hormone (PTH), and, for nearly five decades following his initial hypothesis, scientists pursued the identity of the tumor-borne hypercalcemic factor. Taking advantage of the striking similarities between the biological activities of the Nterminal fragments of PTH and the tumor product, three independent laboratories eventually purified the protein from tumor cells and conditioned media of cultured human lung, breast, renal, and squamous carcinomas (41, 54, 55). Analysis of the N-terminal amino acid sequence of the purified tumor product revealed for the first time that tumors associated with hypercalcemia of malignancy produce a unique gene product that shared limited Nterminal sequence identity with PTH. Because amino acid sequences and biological activities were shared with PTH,this new protein was called parathyroid hormone-related protein (PTHrP). The N-terminal amino acid sequences of F'THrP were employed for the cloning of human PTHrP cDNA (37, 56, 64).the sequences of which were subsequently used to isolate cDNA encoding rat (62, 74), mouse (33, and chicken (50, 63) proteins. As seen in Figure I, 1952

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a comparison of the amino acid sequences of PTHrP from these species demonstrates a remarkably strong phylogenetic conservation of the PTHrP structure, which in turn emphasizes the potential biological importance of this molecule. The sequence identity between the different PTH and PTHrP molecules is highly conserved, yet restricted to the first 13 amino acids. The N-termini of PTHrP and PTH share 8 of the first 13 residues, which lend to their similar biological activities. In vivo, synthetic N-terminal fragments of both PTH and PTHrP are essentially equipotent in their calciotropic properties, including the stimulation of osteoclastic bone resorption (25, 65), renal synthesis of 25-hydroxyvitamin D la-hydroxylase (68). and renal calcium reabsorption (25, 29,

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75). To date, N-terminal peptides of both PTH and F'THrP have been shown to stimulate the same intracellular second messenger systems (12, 16, 42) via their interaction with a common membrane-associated receptor present in traditional (bone and kidney) and nontraditional (e.g., vascular, gastric) PTH target tissues. Although PTHrP also may possibly interact with a unique membrane receptor, at this time, no direct evidence supports the existence of such a molecule. In comparison with human PTHrP, amino acid sequences residues 1 to 110 of the avian and rodent molecules have been well conserved; only 2 and 17 amino acid substitutions are found between the human and rodent or avian molecules, respectively (Figure 1). Simi-

cPRP V T E S P V L 0 N S V T T H N H 1 L R rPRP E D P Q * H T S P T S * S L E P S S * T H hPRP L E G D H L S * T * T * S L E L D S * R H

Figure 1. Comparison of the amino acid sequences of parathyroid hormone-related protein (PTHrP)and parathyroid hormone 0 from chicken (c), rat (r), and human Q. The top line (cPRP) reprcsents the amino acid sequence of cPTHrP that was deduced from a cDNA isolated from a 10-d chicken embryo library (63). This sequence is compared with the corresponding sequences of the rPRP and hPRP PTHrP and the cFTH, r m , and hPTH. Residues that show identity with the cPTWP are designated by an asterisk. A consensus (CUI) sequence for those residues between 1 and 40 that shand by cPTHrP, rF'THrP. and hPTHrP and c m , rPTH, and hPTH is presented on the bottom line. Journal of Dairy Science Vol. 77, No. 7, 1994

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THEDE PTHrP A MultifunctionalGene Product

1

36

PTH-like

Renal Ca2’ reabsorption Osteoclast activity Renal la-hydroxylase Renal + osteoblast adenylyl cylase E I Smooth muscle contractility F ’ Common receptor

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Ca’ * Transport Placenta Breurt 7 Receplor ?

107

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Osleostatin I Bone resorption 1 Osteoclast formation 1 Osteoclast actlvlty

Receptor mediated?

Figure 2. Parathyroid hormone-related protein (PTlirP) encodes at least three bioactive domains. The N-terminal parathyroid hormone O - l i k e domain lies between residues 1 and 34. Residues 67 to 86 have been found to stimulate placental calcium transfer in ovine placenta (11, 33). A pentapeptide termed “osteotstatin” (17, 18), containing residues 107 to 111, is a potent inhibitor of osteoclastogenesis and osteoclast-mediated bone resorption.

lar to the preproopiomelanocortin (POMC) elements and alternatively spliced 3‘gene product, which is proteolytically untranslated exons (36, 73). The human, rat, processed into several bioactive species (M),mouse, and avian PTHrP genes show a PTHrP 1 to 110 contains multiple sites for remarkable conservation of both the organizapotential proteolytic processing of the mole- tion and structure of exon-intron boundaries cple (Figure 2). In support of the possibility (23). Transcription of the PTE-IrP gene is inthat PTHrP can be processed into smaller bio- itiated from up to three independent promoter active peptides, Soifer et al. (53) reported the elements, including a GC-rich “housekeeping” specific intracellular proteolysis and packaging promoter element (67), which is likely responof two peptide products of PTHrP, an N- sible for maintaining the levels of PTHrP terminal 1 to 36 amino acid peptide and a mRNA seen in unstimulated tissues such as the 7-kDa C-terminal fragment. The 7-kDa frag- nonlactating mammary gland (Figure 3). Multiment of PTHrP may have physiological rele- ple AU-rich motifs within the 3’-untranslated vance to calcium translocation because it con- sequence of all known PTHrP mRNA may tains residues 67 to 86, a peptide sequence function in the regulation of PTHrP mRNA shown to stimulate placental calcium transfer stability (51). During the postpartum period, in sheep (11). Proteolysis of PTHrP between this sequence may play an important function residues 106 and 107 may be important for the in the rapid decay of PTHrP mRNA in the release of fragments containing PTHrP 107 to mammary gland following removal of the 111 and PTHrP 107 to 139, which Fenton et suckling stimulus (Figure 3). al. (17, 18) have shown to inhibit osteoclastic The expression and secretion of E’THrP are resorptive activity in vitro. Thus, mounting regulated by numerous factors, including pepevidence indicates that PTHrP possesses a tide and steroid hormones, growth factors, inscope of biological actions beyond the cal- terleukins, vasoconstrictor substances, and biociotropic properties that it shares with PTH. mechanical stretch (58). In vitro and in vivo, Although specific proteolytic fragments of the response of the PTHrP gene to certain PTHrP quite possibly serve important biologi- stimuli generates a transient increase in both cal functions in the mammary gland during PTHrP gene transcription and PTHrP mRNA lactation, direct evidence has not been found accumulation, a response indicative of an early for the physiological production of these frag- response gene (3). Secretion of PTHrP appears ments in vivo. to be coupled with the increase in intracellular The human PTHrP gene is a complex tran- PTNrP mRNA in cultures treated with various scriptional unit containing multiple promoter agents (58), and, during lactation, concentraJournal of Dairy Science Vol. 77, No. 7, 1994

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. U

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Figure 3. Expression of the parathyroid hormone (FfWP) gene in rat mammary gland during pregnancy and the postparmm period. Northern blot analysis of FTHrP mRNA in total RNA (20 pg per lane) prepared from inguinal 7 gland. The PTHrP mRNA in the inguinal mammafy gland obtained on the day of gestation (pest.). at piutunuon @art.), 3 h postparturition @p). or on the designated postpartum day. Postpartum samples were obtained from suckled dams (+) or from dams that w e n not suckled for 4 h (-).

tions of PTHrP secreted into the milk correlate well with the expression of PTHrP mRNA in the mammary gland (71). Unlike PTH, which is essentially a product of the parathyroid gland, PTHrP is produced by numerous cultured cells and tissues. During development, both PTHrP mRNA and PTHrP immunoreactivity are widely distributed in fetal tissues. In the adult, PTHrP is expressed in tissues of the cardiovascular, central, and peripheral nervous systems; gastrointestinal tract; and skin, skeletal muscle, lung, spleen, long bones, kidney, reproductive tissues, urinary bladder, and mammary gland (31, 58). Both PTHrP mRNA and immunoreactivity occur in abnormal as well as normal glandular tissues such as the adrenals, parathyroids and thyroids, pituitary, and pancreas. The expression of PTHrP by keratinocytes, smooth muscle cells, and epithelial cells, including those from the mammary gland, suggests diverse functions for this protein. Over the past 6 yr, great strides have been made to elucidate the potential physiological functions of the N-terminus and other bioactive domains of the PTHrP molecule. For example, evidence now suggests that PTHrP inhibits keratinocyte proliferation because blockade of endogenous PTHrP synthesis via the expression of antisense PTHrP RNA induced proliferation in these cells (26). In view of the potent calciotropic property of PTHrP, regulated production of the molecule by the avian oviduct shell gland of the egg-laying hen (60,61) and the lactating mammary gland sug-

gests that PTHrP may modulate calcium metabolism in these tissues. In support of such an action for PTHrP, investigators have shown that N-terminal (1, 4, 33) and midmolecular (11,33) portions of PTHrP stimulate in a dosedependent manner the transfer of calcium by the ovine placenta. The molecule also appears to encode reciprocal biological activities: the N-terminus, which can stimulate osteoclastic bone resorption (25, 29, 65), and the C terminus, which can inhibit this activity (17, 18). One of the most important bioactivities of PTHrP is likely not related to calcium homeostasis, but is instead related to the contractile activity of vascular and nonvascular smooth muscle. The N-terminal fragments of PTHrP inhibit the contractile activity of smooth muscle in the gut (40),uterus (6, 43, 52), urinary bladder (72), and cardiovascular tissues (58). The production of PTHrP in response to stretching of expandable tissues (13, 59, 72) implicates PTHrP in the regulation of smooth muscle tone. Therefore, PTHrP produced by the lactating mammary gland very likely modulates the activity of mammary epithelial and myoepithelial cells as well as local vascular tissues. Lactating Mammary Gland Production of PTHrP

As shown in Figure 3, the initial evidence to show that PTHrP is a product of the mammary gland was the identification of PTHrP mRNA in the glands of lactating rats (57, 62). Journal of Dairy Science Vol. 77, No. 7, 1994

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High PTHrP mRNA in lactating rat mammary gland contrast with the low concentrations that exist in unsuckled glands or in glands obtained from pregnant animals. As seen in Figure 3, PTHrP mRNA in the mammary gland rise at parturition, and, with suckling, the expression of PTHrP increases further. The link between the synthesis of PTHrP and lactation is emphasized by the short half-life (1 h) of PTHrP mRNA in the gland following the removal of the suckling litter. On postpartum d 1 through 8, PTHrP mRNA in the lactating gland is constant and remains tightly linked to lactation. Removal of the litters for 4 h during this period dramatically reduced PTHrP mRNA content in the gland. Return of the litter for 4 h restored the Concentrations of PTHrP mRNA in the gland. This rapid, reversible expression PTHrP mRNA by the lactating gland was subsequently used to demonstrate that sucklinginduced elevations in serum prolactin stimulated the expression of PTHrP mRNA in the postpartum gland (57). The response to prolactin is specific to the postpartum gland because the dose of prolactin that increased m P in unsuckled postpartum mammary gland failed to alter mammary gland expression of PTHrP mRNA in the tissue during pregnancy. The relative expression of PTHrP mRNA by the rat mammary gland increases between postpartum d 8 and 16; highest expression occurs in the tissue at weaning (Figure 3). The accumulation of PTHrP mRNA in the lactating mammary gland at this time is also more stable because the removal of the suckling litter for 4 h failed to reduce PTHrP mRNA in the gland. The relative increase in the concentrations of PTHrP mRNA in the mammary gland during this time is reflected in higher milk content of PTHrP (71) and may be due to the temporal increase in the demand for milk. Therefore, the expression of PTHrP by the mammary gland is tightly linked to lactation but is also differentially regulated by the gland during the postpartum period. The physiological significance of the relative increase in PTHrP mRNA abundance and the apparent increase in message half-life during the week before weaning remains to be determined. Using both immunohistochemistry and in situ hybridization, Rakopoulos et al. (45) investigated the cellular localization of immunoreactive PTHrP and PTHrP mRNA synJournal of Dairy Science Vol. 77, No. 7, 1994

thesis in the rat mammary gland during pregnancy and lactation. In those studies, both the antigen and mRNA were found to localize to epithelial cells lining alveoli in tissues of both pregnant and lactating animals, suggesting a role for PTHrP in the development and subsequent function of the gland. To support those findings, PTHrP was shown to be synthesized by cultures of mammary gland epithelial cells (19). Secretion of PTHrP into Milk

Following the initial identification of the PTHrP structure from tumor cells, radioimmunoassays were developed for the detection of immunoreactive PTHrP in blood (8, 9, 10, 20, 46). Such assays were also employed to examine whether PTHrP was present in milk of various species. In their initial study, Budayer et al. (8) determined that PTHrP was present in milk at concentrations 5 to 10,000 times those measured in plasma. The results of other studies (20, 46) have now confirmed the presence of PTHrP in milk, and several laboratories (46,66) have now purified milk PTHrP to show that the molecular mass of the milk proteins reflected molecules smaller than PTHrP 1 to 141. The ability of milk F'THrP to bind and to activate second messenger systems of the PTH receptor complex indicates that the biologically active N-terminus of the milk protein remains intact. As seen in Figure 3, expression of PTHrP mRNA by the lactating rat mammary gland increases during the week before weaning. Yamamoto et al. (70) demonstrated that this temporal increase in PTHrP mRNA is reflected in higher concentrations of immunoreactive and bioreactive PTHrP in rat milk. Studies have now shown a lack of correlation between the concentrations of calcium and immunoreactive PTHrP in bovine and rat milk (20, 45, 71). Potential Biological Roles for PTHrP

The initial discovery that mammary gland production of PTHrP was tightly linked to lactation suggested the exciting possibility that this newly discovered calcium-mobilizing protein may be an important systemic factor linking lactogenesis to renal and bone calcium homeostasis (62). In the years that followed, some, but not all, studies generated evidence to

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support such a function for the protein. Secretion of relatively high quantities of bioactive PTHrP into the milk suggests a possible, yet undetermined function for the protein in neonatal development. Recent studies indicate that PTHrP produced by the gland may act directly on local mammary gland physiology. Figure 4 summarizes current understanding of the potential roles for PTHrP during lactation.

homeostasis. They found that this treatment had no measurable effect on serum calcium or the calcium content in neonatal bone. In this same study, the subcutaneous, but not oral administration, of PTHrP 1 to 34 increased serum calcium, indicating that the peptide in milk may not accumulate in the circulation to the concentrations required to alter calcium metabolism. Goff et al. (20) reported that immunoreactive PTHrP was elevated in the plasma of newborn calves following colostrum Role in Neonatal Development feeding. Although plasma concentrations of Milk is very abundant in bioactive F"HrP, immunoreactive PTHrP were clearly elevated being up to 5 to 10,OOO times the amount in following feeding, the PTHrP in neonatal blood (8, 20, 22, 27, 30, 46).This abundance plasma was not biologically active in assays suggests either that high concentrations of that measured N-terminal bioactivity. Perhaps PTHrP are required to act on local mammary these data were somewhat expected because tissues or that the milk PTHrP serves a func- neonatal calcium is not generally elevated foltional significance in the neonate. To address lowing nursing. Other bioactive regions the latter point, Kukreja et al. (32) injected (Figure 2) within the PTHrP structure, aside anti-FTHrP antiserum into neonatal mice to from the N-terminal PTH-like domain, may examine the effect that neutralization of cir- possibly possess activities as yet to be deterculating PTHrP would have on calcium mined in neonatal circulation.

Q

Neonatal Development A. 1 GI ~ i l l l t y B. I GI absorption of Ca2* C. t Systemic circulation

Local Blood Flow

f

A. I Vascular resistance B. I Vasoconstrictor synthesis

b

MAMMARY OUND PTHrP

Endocrlne A. t Renal Ca'+ reabsorption E. I Osieoclastlc bone resorptlon C. I Gut absorption of Ca"

Autocrine-Paracrine A. 1 Metabolic actkiiy 6. Caz+translocation C. I Secretion

Figure 4. Potential functions for parathyroid hormone-related protein (PI")produced by the mammary gland during lactation. GI = Gastrointestinal. Journal of Dairy Science Vol. 77, No. 7, 1994

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goats. In those studies, lactation was associated The calciotropic property of PTHrP, to- with a measurable increase in PTHrP 1 to 86 gether with the tight regulation of its synthesis in blood obtained from the mammary gland by the mammary gland during lactation, sug- vein during the early postpartum period. Measgests that PTHrP may be a calcium-mobilizing urable elevations of PTHrP in venous plasma endocrine factor released into the systemic cir- were blocked by treatment with bromocrypculation during lactation. The PTHrP released tine, suggesting that the release of PTHrP into into the circulation during suckling could inter- the venous blood is dependent on serum act with receptors in bone and kidney to in- prolactin. These results suggest that the failure crease the movement of calcium into blood. of others (8, 30) to identify elevations in Interestingly, even before it was structurally PTHrP in plasma from forearm veins may be identified as PTHrP, Brommage and DeLuca due to the rapid clearance of PTHrP in plasma. (7) suggested that the tumoral hypercalcemic Whether the PTHrP released during lactation factor may be involved in this process. Once has any direct consequences on calcium metabsensitive radioimmunoassays for PTHrP were olism of bone and kidney remains to be deterdeveloped, the concentrations of PTHrP in the mined. blood of nursing women and animals could be assessed. Initial studies (8, 30) showed that Local Transfer of Calcium PTHrP concentrations in forearm venous blood Although synthetic N-terminal peptides of were generally very low and did not change PTHrP stimulate the reabsorption of renal calfollowing the initiation of suckling, indicating cium and release of calcium from bone, the that the mammary gland does not release first evidence implicating PTHrP in the transmeasurable amounts of PTHrP into blood. Furlocation of calcium across biological memther evidence not in support of an endocrine branes was presented by Rodda et al. (49). function for PTHrP during lactation was Using an ovine model of placental calcium presented by Melton et al. (38) in their studies of lactating mice. In their studies, passive im- transfer, they showed that both synthetic munization to PTHrP via the injection of an PTHrP and PTHrP extracted from the fetal anti-PTHrP antiserum into lactating mice was parathyroid glands and placenta could restore used to show that PTHrP neutralization did not the maternal-fetal calcium gradient that was alter maternal calcium, milk calcium, or pup lost as a result of fetal thyroidparathyroidectomy. These initial findings, weight gain. Two studies have recently compiled indirect along with the data from a recent study by and direct evidence in support of an endocrine Barlet et al. (4), suggest that the N-terminus of function for PTHrP. Yamamoto et al. (70) stud- PTHrP possesses a portion of this activity. ied the effects of lactation on renal function Experimental results of Care et al. (11) and and found that the initiation of lactation was MacIsaac et al. (33) now strongly suggest that associated with transient increases in renal ex- this activity is encoded in PTHrP 67 to 86 and cretion of phosphate and cyclic AMP. They not PTHrP 1 to 34 (Figure 2). Whether PTHrP found that these responses were similar to that 67 to 86 interacts with the same plasma memgenerated following a single injection of brane receptor as the N-terminus of PTHrP PTHrP 1 to 34 and could be measured in remains to be determined. In keeping with the model described, Figure lactating rats following removal of their parathyroid glands. Additional indirect evi- 5 suggests the possible autocrine or paracrine dence for endocrine activity for PTHrP during actions of PTHrP on mammary epithelial cells lactation has been provided in preliminary data during lactogenesis. In view of the ability of by Miller et al. (39) in their study that showed PTHrP to stimulate placental calcium transfer, a correlation between the increase in bone Barlet et al. (5)studied the effect of synthetic resorption and the high concentrations of PTHrP fragments on calcium secretion into PTHrP synthesis during early lactation. The milk during early lactation in the goat and only direct evidence to support release of found that PTHrP 1 to 34 and PTHrP 1 to 86, PTHrP by the gland during lactation has been but not PTHrP 140 to 173, increased the calprovided by Ratcliffe et al. (47), using lactating cium, magnesium, and inorganic phosphate Journal of Dairy Science Vol. 77, No. 7, 1994

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content in milk in a time-dependent manner. Regulation of Mammary Gland Hemodynamics None of these peptides altered milk production In addition to the calciotropic actions of during the experiment. These data support an PTHrP, infusion of N-terminal fragments of association between the concentrations of the protein can increase cardiac output and PTHrP in milk and the secretion of minerals regional blood flow (48) via their interaction into milk. However, the arterial hypercalcemia induced by infusion of F'THrP 1 to 34 and with receptors present in the heart, aorta, and PTHrP 1 to 86 suggests that the action of peripheral vascular tissues. The vasodilatory PTHrP delivered in this manner may not be action of PTHrP on peripheral blood vessels directed solely to the mammary epithelium, results from the ability of the peptide to inhibit but may also alter systemic calcium homeosta- the contractile activity of vascular smooth sis. Because PTHrP 67 to 86 stimulates muscle (61,69). The production of both PTHrP placental mineral transfer but does not induce and its receptor throughout the cardiovascular hypercalcemia, it is important to examine the system and in cultured vascular smooth muscle effect of this midmolecular fragment of PTHrP cells (24, 44) indicates that PTHrP and its on mineral secretion by the mammary gland. receptor represent an autocrine-paracrine ac-

Milk

Plasma

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Epithelium Figure 5. Parathyroid hormone-dated protein (PTHrF') may facilitate the translocation of calcium from serum to milk by mammary epithelial cells. The PTHrP produced by epithelial cells may interact with receptors on the surface of either the cell or adjacent cells. Because FTHrP is concentrated in the milk, the protein may possibly have never left the cell but acted on signaling pathways present on the cytoplasmic face of the plasma membrane or within other intracellular comparbnents of the expressing cell.

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tivity. Furthermore, the increased PTHrP synthesis and secretion by vascular smooth muscle cells by a number of vasoconstrictor substances (44)further emphasize the potentially important role for F'THrP in the regulation of vascular tone. Lactation is associated with increased cardiac output and blood flow to the mammary gland and other tissues (21). The presence of PTHrP and its receptor in the nutrient vasculature of the rat inguinal mammary gland and the regulation of ETHrP mRNA during lactation (60,62) support a potential role for the protein in the regulation of mammary gland hemodynamics. Recently, Davicco et al. (15) presented evidence to support a direct role for PTHrP in increasing blood flow to the mammary gland of sheep. Using flow probe analysis, they found that infusion of PTHrP 1 to 34 into the mammary artery rapidly increased blood flow to the gland in a dose-dependent manner. The local action of the infused peptide was supported by the localization of the response to the infused gland and the lack of hypercalcemia following peptide infusion. Thus, PTHrP can quite possibly modulate blood flow to the mammary gland via its local expression and action in both the nutrient vasculature as well as vascular beds of the gland. ~iucuruion

It has been approximately 5 yr since E'THrP was first shown to be produced by the lactating mammary gland. During this time, the results of numerous studies have advanced the understanding of the potential physiological function that the protein might play during lactation. Although a small body of evidence supports an endocrine activity for the protein, most do not; thus, further studies are needed to determine whether the PTHrP produced by the mammary gland is responsible for the responses measured in kidney (70) and bone (39). The accumulation of evidence in support of a local action of PTHrP in cells and tissues supports the view that PTHrP likely modulates local activities of the mammary gland, including mineral secretion and gland blood flow during lactation. Although studies have shown that PTHrP present in skin (14) can regulate proliferation and differentiation of cultured cells, such as keratinocytes (26), the effects of ETHrP on mamJournal of Dairy Science Vol. 77, No. 7, 1994

mary gland epithelial cell proliferation or function have not been reported. The presence of relatively high concentrations of PTHrP in milk suggests that this protein may function in neonatal development; however, positive experimental results to support such a role for PTHrP in neonatal calcium homeostasis have not been forthcoming. A powerful in vivo model to test the importance of PTHrP in mammary gland biology would be a genetically modified mouse in which the F'THrP gene is rendered nonfunctional via homologous recombination. Such a mouse has recently been engineered (28), but unfortunately, the homozygous offspring die at birth. These results emphasize the biological importance of this gene product, but the limited lifespan of these mice makes it impossible to determine the effect or effects that this gene removal will have on the lactating mammary gland. An alternative approach would be to engineer a mouse that expresses the antisense RNA in the mammary gland via a tissuespecific promoter element. Such an approach has been successful in vitro to demonstrate the anti-proliferative action of the protein in keratinocytes (26). The continued application of creative approaches to this field of study will result in an even greater understanding of the biological role of this calciotropic protein in mammary gland biology in the next 5 yr. ACKNOWLEDGMENTS

I thank Melissa Thlede, Ellen Thiede, Donna Petersen, Bill Grasser, and Steve Smock for help in the preparation of this manuscript. REFERENCES 1 Abbas, S. K., D. W. Pickard, C. P. Rodda. J. A. Heath, R. G. Hammonds. W. I. Wood, I. W. Caple. T. J. Martin, and A. D. Can.1989. Stimulation of ovine placental calcium transpoa by purified natural and recombinant parathyroid hormone-related protein (PTHrP) preparations. I. Exp. Physiol. 74549. 2 Albright, E 1941. Case records of the Massachusetts General Hospital. Case 27461. New England J. Med. 225:789. 3 Allinson, E. T., and D. J. Drucker. 1992. Parathyroid hormone-like peptide shares features with members of the early response gene family: rapid induction by SNIII, growth factors, and cycloheximide. Cancer Res. 52:3103. 4Barlet, J.-P., C. Champdon, V. Coxam, M. J. Davicco, and J. C. Tressol. 1992. Parathyroid

SYMPOSIUM: CALCIUM METABOLISM AND UTILIZATION hormone-related peptide might stimulate calcium secretion into the milk of goats. J. Endocrinol. 132:

353. 5 Barlet. J.-P., M. J. Davicco, and V. Coxam. 1990. Synthetic parathyroid hormone-related Peptide(1-34) fragment stimulates placental calcium transfer in ewes. J. Endocrinol. 127:33. 6Barri. M. E.. A. B. Abbas. and A. D. Care. 1992. The effects in the rat of two fragments of parathyroid hormone-related protein on uterine contractions in situ. Exp. Physiol. 77481. 7Brommagc. R., and H. F. DeLuca. 1985. Regulation of bone mineral loss during lactation. Am. J. Physiol. 248:E182. 8 Budayr, A. A., B. P. Halloran. J. C. King, D. Diep, R. A. Nissenson, and G. J. Strewler. 1989. High levels of a parathyroid hormone-lile protein in milk. Roc.Natl. Acad. Sci. USA 86:7183. 9Budayr, A. A,, R. A. Nissenson, R. F. Klein, K. K. Pun, G. H. Clark, D. Diep, C. D. b a u d , and G. J. Strewler. 1989. Increased serum levels of a parathyroid hormone-like protein in malignancyassociated hypercalcemia. Ann. Intern. Med. 111:807. 10 Burtis, W. J., T. G. Brady, J. J. Orloff, J. B. Ersbak, R. P. Warrell. T. L. Olson, T. L. Wu. M. E. Mitnick, A. E. Broadus, and A. F. Stewart. 1989. Immunochemical characterization of circulating parathyroid hormone-related protein in patients with humoral hypercalcemia of cancer. New Eagland J. Med. 322: 2206. 11 Care, A. D., S. K. Abbas, D. W. Pickard. M. Bani, M. Drinkhill. J. B. Findlay, I. R. White, and I. W. Caple. 1990. Stimulation of ovine placental traasport of calcium and magnesium by mid-molecule fragments of human parathyroid hormone-related protein. J. Exp. Physiol. 75605. 12 Civitelli. R., T. J. Martin, A. Fausto. S. L. Gunsten, K. A. Hruska, and L. V. Avioli. 1989. Parathyroid hormone-related peptide transiently increases cytosolic calcium in osteoblast-like cells: comparison with parathyroid hormone. Endocrinology 125:1204. 13 Daifotis, A. G., E. C. Weir, B. E. Dreyer. and A. E. Broadus. 1992. Stretch-induced parathyroid hormonerelated peptide gene expression in the rat uterus. J. Biol. Chcm. 267:23,455. 14Danks. J. A., P. R. Ebeling, J. Hayman, S. T. Chou, J. M. Moseley, J. Dunlap. B. E. Kemp, and T. J. Martin. 1989. Parathyroid hormone-related protein: immunohistochernicd localization in cancers and in n o d skin. J. Bone Miner. Res. 4:273. 15 Davicco, M.-J., D. D m d , J. Lefaivre, and J.-P. Barlet. 1993. Parathyroid hormone-related protein might increase m a m m y blood flow.J. Bone Miner. Res. 8:1519. 16 Donahue, H. J.. M. J. Fryer, and H. Heath. 111. 1990. Structure-function relationships for full-length recombinant parathyroid hormone-related peptide and its amino-terminal fragments: effects on cytosolic calcium ion mobilization and adenylate cyclase activation in rat osteoblast-like cells. Endocrinology 126: 1417. 17 Fenton. A. J., B. E. Kemp, R. G. Hammonds, K. Mitchelhill. J. M. Moseley, T. J. Martin, and G. C. Nicholson. 1991. A potent inhibitor of osteoclastic bone resorption within a highly conserved pentapep-

1961

tide region of parathyroid hormone-related protein; PTHrP (107-111). Endocrinology 129:3424. 18 Fenton. A. J., B. E.Kemp, G. N. Kent, J. M. Moseley, M. H.Zheng, D. J. Rowe, J. M. Britto, T. J. Martin, and G. C. Nicholson. 1991. A carboxyl-terminal peptide from the parathyroid hormone-related protein inhibits bone resorption by osteoclasts. Endocrinology 1291762. 19FerrSri, S. L., R. Riuoli, and J.-P. Bonjour. 1992. Parathyroid hormone-related protein production by primary culhlres of mammary epithelial cells. J. Cell Physiol. 150304. 20Goff. J. P., T. A. Reinhardt. S. Lee, and B. W. Hollis. 1991. Parathyroid hormone-related peptide content of bovine milk and calf blood assessed by radioimmunoassay and bioassay. Endocrinology 129:2815. 21 Hanwell, A., and J. L. Linzell. 1973. The time course of cardiovascular changes in lactation in the rat. J. Physiol. (Land.) 233:93. 22Henderson. J. E., C. Shustik, R. Kremer. S. A. Rabbani, G. N. Hendy. and D. Goitman. 1990. Circulating concentrations of parathyroid hormone-related peptide in malignancy and in hyperparathyroidism. J. Bone Miner. Res. 5:105. 23Hendy. G. N.. and D. Goltzman. 1992. Molecular biology of parathyroid hormone-like peptide. Page 25 in Parathyroid Hormone-Related Protein: Normal Physiology and Its Role in Cancer. B. P. Halloran and R. A. Nissenson, ed. CRC Ress, Boca Raton, FL. 24Hongo. T.. J. Kupfer, H. Enomoto, B. S h d i , D. Giiannella-Neto, J. S. Fomster, F. R. Singer. D. Goltwnan, G. N. Hendy, 1. A. Fagin, and T. L. Clemens. 1991. Abundant expression of parathyroid hormone-related protein in primary rat aortic smooth muscle cells accompanies serum-induced proliferation. J. Clin. Invest. 88:1841. 25Horiuchi. N., M. P. Caulfield, J. E. Fisher, M. E. Goldman, R. L. McKee, J. J. Levy, R. F. Nutt, S. B. Rodan, T. L. Schofield, T. L. Clemens. and M. Rosenblatt. 1987. Similarity of synthetic peptide from human tumor to parathyroid hormone in vivo and in vim. Science 238:1566. 26 Kaiser, S. M., P. Laneuville, S. M. Bernier, J. S.Shim, R. Kremer. and D. Goltman. 1992. Enhanced growth of a human keratinocyte cell line induced by antisense RNA for parathyroid hormone-related peptide. J. Biol. Chem. 267:13.623. 27 Kao, P. C.. G. G. Klee, R. L. Taylor, and H. Heath, In. 1990. Parathyroid hormone-related peptide in plasmas of patients with hypercalcemia and malignant lesions. Mayo Clin. Proc. 65:1399. 28Karaplis. A,, V. Tybulewicz, R. Mulligan and H. Kronenberg. 1992. Disruption of parathyroid hormone-related peptide gene leads to a multitude of skeletal abnormalities and perinatal mortality. J. Bone Miner. Res. (Suppl. 1): Al. ( A b . ) 29Kemp. B. E., J. M. Moseley, C. P. Roada P. R. Ebehg, R.E.H. Wettenhall, D. Stapleton, H. Diefenbach-Jagger, F.Ure. V. P. Michelangeli. H. A. SimmOna L. G. Raisz, and T. J. Martin. 1987. Parathyroid hormone-related protein of malignancy: active synthetic fragments. Science 238:1568. 30Khosla, S.,K. L. Johanson, S. J. Ory, P. C. O’Brien, and P. C. Kao. 1990. Parathyroid hormone-related Journal of Dairy Science Vol. 77, No. 7, 1994

1962

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peptide in lactation and in umbilical cord blood. Mayo Clin. Proc. 65:1408. 31 Kramer, S.,F. H. Reynolds, M. Castillo. D. M. Valenzuela, M. Thorikay, and J. M. Sorvillo. 1991. Immunological identification and distribution of parathyroid hormone-lie protein polypeptides in normal and malignant tissues. Endocrinology 128:1927. 32Kubeja, S. C., J. J. DAnza, M. E. Melton, S. A. Wimbiscus, V. Grill, and T. J. Martin. 1991. Lack of effects of neutralization of parathyroid hormonerelated protein in calcium homeostasis in neonatal mice. J. Bone Miner. Res. 6:1197. 33MacIsaac. R. I.. J. A. Heath, C. P. Rodda, J. M. Moseley, A. D. Care, T. J. Martin, and I. W. Caple. 1991. Role of the fetal parathyroid glands and parathyroid hormone-related protein in the regulation of placental transport of calcium magnesium, and inorganic phosphate. Reprod. F e d . Dev. 3:447. 34Mains. R. E., and B. A. Eipper. 1990. The tissuespecific processing of pro-ACTWendorphin-recent advances and unsolved problems. Trends Endocrinol. Metab. 1:388. 35Mangin. M., K. Ikeda, and A. E. Broadus. 1990. Structure of the mouse gene encoding parathyroid hormone-related peptide. Gene 95: 195. 36Mangin, M., K. Ikeda, B. E. Dreyer, and A. E. Broadus. 1989. Isolation and characterization of the human parathyroid hormone-like peptide gene. Roc. Natl. Acad. Sci. USA 86:2408. 37 Mangin, M., A. C. Webb, B. E. Dreyer, J. T. Posillico, K. Ikeda, E. C. Weir, A. F. Stewart, N. H. Bander, L. Milstone. D. E. Barton, U. Franke. and A. E. Broadus. 1988. Identification of a cDNA encoding a parathyroid hormone-like peptide from a human tumor associated with hypercalcemia of malignancy. Proc. Natl. Acad. Sci. USA 85597. 38 Melton, M. E.. J. J. DAnza, S. A. Wimbiscus. V. Grill, T. J. Martin, and S. C. Kulrreja. 1990. Parathyroid hormone-related protein and calcium homeostasis in lactating mice. Am. I. Physiol. 259: E792. 39 Miller, S. C., C. M. Bagi, and B. M. Bowman. 1991. Increased serum levels of parathyroid hormone-related peptide correlate with the activation of bovine resorption and remodelling during early lactation in rats. Calcif. Tissue Int. 48: (Suppl. 1) A2. ( A b . ) 40 Mok, L.L.S.,C. W. Cooper, and J. C. Thompson. 1989. Parathyroid hormone and parathyroid honnonerelated protein inhibit phasic contraction of pig duodenal smooth muscle. Roc. Soc. Exp. Biol. Med. 1991:337. 41 Moseley, J. M., M. Kuboto. H. Diefenbach-Jagger, R.E.H. Wettenhall, B. E. Kemp. L. J. Suva, C. P. Rodda, P. R. Ebeling, P. J. Hudson, J. D. Zajac, and T. J. Martin. 1987. Parathyroid-related protein purified from human lung cancer cell line. Roc. Natl. Acad. Sci. USA 84:5048. 42 Nissenson. R. A,, D. Diep, and G. J. Strewler. 1988. Synthetic peptides comprising the amino-terminal sequence of a parathyroid hormone-like protein from human malignancies. Binding to parathyroid hormone receptors and activation of adenylate cyclase in bone cells and kidney. J. Biol. Chem. 263:12,866. 43 Paspaliaris, V., S. J. Vargas, M. T. Gillespie. E. T. Williams, J. A. Danks, J. M. Moseley, M. E. Story, J. Journal of Dairy Science Vol. 77, No. 7. 1994

N. Pennefather, D. D. Leaver, and T. J. Martin. 1992. Oestrogen enhancement of the myometrial response to exogenous parathyroid hormone-related protein (PTHrP),and tissue localization of endogenous PTHrP and its mRNA in the virgin uterus. J. Endocrinol. 134: 415. 44Pirolq C. J., H. Wang, A. Kamyar, S. Wu, H. Enomoto, B. Sharifi, J. S. Forrester, T. L. Clemens, and J. A. Fagin. 1993. Angiotensin I1 regulates parathyroid hormone-related protein expression in cultured rat aortic smooth muscle cells through transcriptional and post-transcriptional mechanisms. J. Biol. Chem. 268:1987. 45 Rakopoulos. M., S. J. Vargus, M. T. Gillespie, P.W.M. Ho, H. Diefenbach-Jagger, D. D. Leaver, V. Grill, J. M. Moseley, J. A. Danks, and T. J. Martin. 1992. Production of parathyroid hormone-related protein by the lactating mammary gland in pregnancy and lactation. Am. I. Physiol. 263:ElM7. 46Ratcliffe, M. A., E. Green, J. Emly, S. Norbury, M. Linsay, D. A. Heath, and J. G. Ratcliffe. 1990. Identification and partial characterization of parathyroid hormone-related protein in human and bovine milk.J. Endocrinol. 127:167. 47 Ratcliffe, W. A., G. E. Thompson, A. D. Care, and M. Peaker. 1992. Production of parathyroid hormonerelated protein by the mammary gland of the goat. J. Endocrinol. 133:87. 48 Roca-Cusachs, A,, D. I. Dipette, and G. A. Nickols. 1991. Regional and systemic hemodynamics effects of parathyroid hormone-related protein: preservation of cardiac function and coronary and renal flow with reduced blood pressure. J. Pharmacol. Exp. Ther. 256: 110. 49 Rodda, C. P., M. Kubota. J. A. Heath, P. R. Ebeling, J. M. Moseley, A. D. Care, I. W. Caple, and T. J. Martin. 1988. Evidence for a novel parathyroid hormone-related protein in fetal parathyroid glands and sheep placenta: comparisons with a similar protein implicated in humoral hypercalcemia of malignancy. J. Endocrinol. 117:261. 50Schermer, D. T., S.D.H. Chan, R. Bruce, R. A. Nissenson, W. I. Wood, and G . J. Strewler. 1991. Chicken parathyroid hormone-related protein and its expression during embryologic development. 1. Bone Miner. Res. 6:149. 51 Shaw, G., and R. A. Kamen. 1986. A conserved AU sequence from the 3’-untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46:659. 52Shew, R. L., J. A. YE, D. B. Kliewer, Y. J. Keflemariam, and D. L. McNeill. 1991. Parathyroid hormone-related protein inhibits stimulated uterine contraction in vitro. 1. Bone Miner. Res. 6:955. 53 Soifer, N. E., K. E. Dee, K. L. Insogna, W. J. Bwtis, L. M. Matovik, T. L. Wu, L. M. Milstone, A. E. Broadus, W. M. Philbrick, and A. F. Stewart. 1992. Parathyroid hormone-related protein: evidence for secretion of a novel mid-region fragment by three different cell types. J. Biol. Chem. 267:18,236. 54Stewart. A. F., T. Wu. D. Goumas, W. J. Burtis, and A. E. Broadus. 1987. N-Terminal amino acid sequence of two novel tumor-derived adenylate cyclasestimulating proteins: identification of parathyroid

SYMPOSIUM: CALCIUM METABOLISM AND UTILIZATION hormone-like and parathyroid-unlike domains. Biochem. Biophys. Res. Commun. 146:72. 55 Strewler, G. J., P. H. Stem, J, W. Jacobs, J. Eveloff, R. F. Klein, S. C. hung, M. Rosenblatt, and R. A. Nissenson. 1987. Parathyroid hormone-like protein from human renal carcinoma cells. Structural and function homology with parathyroid hormone. J. Clin. Invest. 80 1803. 56 Suva, L. J., G. A. Winslow, R.E.H. Wettenhall, R. G. Hammonds, J. M. Moseley, H. Diefenbach-Jagger, C. P. Rod& B. E. Kemp, H. Rodriguez, E. Y. Chen, P. J. Hudson, T. J. Martin, and W. 1. Wood. 1987. A parathyroid hormone-related protein implicated in malignant hypercalcemia: cloning and expression. Science 237:893. 57Thiede. M. A. 1989. The mRNA encoding a parathyroid hormone-like peptide is produced in mammary tissue in response to elevations in serum prolactin. Mol. Endocrinol. 3:1443. 58 Thiede. M. A. 1992. Expression and regulation of the parathyroid hormone-related protein gene in tumors and n o d tissues. Page 57 in Parathyroid HormoneRelated Protein: Normal Physiology and Its Role in Cancer. B. P. Halloran and R. A. Nissenson, ed. CRC Press, Boca Raton, FL. 59Thiede. M. A., A. G. Daifotis, E. C. Weir, M. L. Brines, W. J. Butis, K. Ikeda, B. E. Dreyer, R. E. Garfield, and A. E. Broadus. 1990. Intrauterine occupancy controls expression of the parathyroid hormone-related peptide gene in preterm rat myometrium. Proc. Natl. Acad. Sci. USA 87:6%9. 60Thiede, M. A.. W. A. Grasser, and D. N. Petersen. 1992. Regulated expression of parathyroid hormonerelated protein in mammary blood supply supporn a role in mammary blood flow.Bone Miner. 17 (Suppl. 1) A8. (AM.) 61Thiede. M. A., S. C. Harm, R. L. McKee, W. A. Grasser, L. T. Duong, and R. M. Leach. 1991. Expression of the parathyroid hormone-related protein gene in the avian oviduct: potential role as a local modulator of vascular smooth muscle tension and shell gland motility during the egg-laying cycle. Endocrinology 129:1958. 62 Thiede, M. A., and G. A. Rodan. 1988. Expression of a calcium-mobilizing parathyroid hormone-like p e g tide in lactating mammary tissue. Science 242:278. 63Thiede. M.A., and S. J. Rutledge. 1990. Nucleotide sequence of a parathyroid hormone-related peptide by the 10 day chicken embryo. Nucleic Acids Res. 18: 3064. 64Thiede, M.A.. G. I. Strewler, R. A. Nissenson, M. Rosenblatt, and G. A. Rodan. 1988. Human renal carcinoma expresses two messages encoding a parathyroid hormone-like peptide: evidence for the

1963

alternative splicing of a single copy gene. Proc. Natl. Acad. Sci. USA 85:4605. 65 Thompson, D. D., I. G. Seedor, J. E. Fisher, M. Rosenblatt, and G. A. Rodan. 1988. Direct action of the parathyroid hormone-like human hypercalcemic factor on bone. Proc. Natl.Acad. Sci. USA 85:5673. 66Thurston, A. W., J. A. Cole, L. S. Hillman, J. H. lm, P. K. Thome, W. J. Krause, J. R. Jones, S . L. Eber, and L. R. Folte. 1990. Purification and properties of parathyroid hormone-related peptide isolated from milk. Endocrinology 126:1183. 67 Vasavada R., J. J. Wysolmerski, A. E. Broadus, and W. M. Philbrick. 1993. Identification and characterization of a GC-rich promoter of the human parathyroid hormone-related protein gene. Mol. Endocrinol. 7:273. 68 Walker, A. T., A. F. Stewart, E. A. Kom, T. Shiratori, M. A. Mitnick, and T. 0. Carpenter. 1989. Effect of parathyroid hormone-lie peptides on 1.25hydroxyvitamin D-1-alpha-hydroxylase activity in rodents. Am. J. Physiol. 256:E309. 69 Winquist, R. J., E. P. Baskin, and G. P. Vlasuk. 1987. Synthetic tumor-derived human hypercalcemic factor exhibits parathyroid hormone-like vasorelaxation in renal arteries. Biochem. Biophys. Res. Commun. 149: 227. 70Yamamoto. M.,L. T. Duong, J. E. Fisher, M. A. Thi&, M. P. Caulfield, and M. Rosenblatt. 1991. Suckling-mediated increases in urinary phosphate and 3’,5’-cyclic adenosine monophosphate excretion in lactating rats: possible systemic effects of parathyroid hormone-related protein. Endocrinology 129:2614. 71 Yamamoto, M., J. E. Fisher, M. A. Thiede, M. A. Caulfield, M. Rosenblatt. and L. T. Duong. 1992. Concentration of parathyroid hormone-related protein in rat milk change with the duration of lactation and interval from previous suckling, but not with milk calcium. Endocrinology 130:741. 72 Yamamoto. M., S. C. Harm, W. A. Grasser, and M. A. Thiede. 1992. Parathyroid hormone-related protein in the rat urinary bladder: a smooth muscle relaxant produced locally in response to mechanical stretch. Roc. Natl. Acad. Sci. USA 895326. 73 Yasuda, T., D. Banville, G. N. Hendy, and D. Goltzman. 1989. Characterization of the human parathyroid hormone-like peptide gene. J. Biol. Chem. 264:7720. 74Yasuda, T., D. Banville, S. A. Rabbani, G. N. Hendy and D. Goltwnan. 1989. Rat parathyroid hormone-like peptide: comparison with the human homologue and expression in malignant and normal tissue. Mol. Endocrinol. 3518. 75Zhou. H., D. D. haver, J. M. Moseley, B. Kemp, P. R. Ebeling, and T. J. Martin. 1989. Actions of parathyroid hormone-related protein on the kidney in vivo. J. Endocrinol. 122:229.

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