Increased expression of COX-2 or VEGF-C has been correlated with progressive disease in certain cancers. Present study utilized several human breast cancer cell lines (MCF-7, T-47D, Hs578T and MDA-MB-231, varying in COX-2 expression) as well as 10 human breast cancer specimens to examine the roles of COX-2 and prostaglandin E (EP) receptors in VEGF-C expression or secretion, and the relationship of COX-2 or VEGF-C expression to lymphangiogenesis. We found a strong correlation between COX-2 mRNA expression and VEGF-C expression or secretion levels in breast cancer cell lines and VEGF-C expression in breast cancer tissues. Expression of LYVE-1, a selective marker for lymphatic endothelium, was also positively correlated with COX-2 or VEGF-C expression in breast cancer tissues. Inhibition of VEGF-C expression and secretion in the presence of COX-1/2 or COX-2 inhibitors or following downregulation of COX-2 with COX-2 siRNA established a stimulatory role COX-2 in VEGF-C synthesis by breast cancer cells. EP1 as well as EP4 receptor antagonists inhibited VEGF-C production indicating the roles of EP1 and EP4 in VEGF-C upregulation by endogenous PGE 2 . Finally, VEGF-C secretion by MDA-MB-231 cells was inhibited in the presence of kinase inhibitors for Her-2/neu, Src and p38 MAPK, indicating a requirement of these kinases for VEGF-C synthesis. These results, for the first time, demonstrate a regulatory role of COX-2 in VEGF-C synthesis (and thereby lymphangiogenesis) in human breast cancer, which is mediated at least in part by EP1/EP4 receptors.

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Stanniocalcin (STC)-2 was discovered by its primary amino acid sequence identity to the hormone STC-1. The function of STC-2 has not been examined; thus we generated two lines of transgenic mice overexpressing human (h)STC-2 to gain insight into its potential functions through identification of overt phenotypes. Analysis of mouse Stc2 gene expression indicates that, unlike Stc1, it is not highly expressed during development but exhibits overlapping expression with Stc1 in adult mice, with heart and skeletal muscle exhibiting highest steady-state levels of Stc2 mRNA. Constitutive overexpression of hSTC-2 resulted in pre-and postnatal growth restriction as early as embryonic day 12.5, progressing such that mature hSTC-2-transgenic mice are ϳ45% smaller than wild-type littermates. hSTC-2 overexpression is sometimes lethal; we observed 26 -34% neonatal morbidity without obvious dysmorphology. hSTC-2-induced growth retardation is associated with developmental delay, most notably cranial suture formation. Organ allometry studies show that hSTC-2-induced dwarfism is associated with testicular organomegaly and a significant reduction in skeletal muscle mass likely contributing to the dwarf phenotype. hSTC-2-transgenic mice are also hyperphagic, but this does not result in obesity. Serum Ca 2ϩ and PO4 were unchanged in hSTC-2-transgenic mice, although STC-1 can regulate intra-and extracellular Ca 2ϩ in mammals. Interestingly, severe growth retardation induced by hSTC-2 is not associated with a decrease in GH or IGF expression. Consequently, similar to STC-1, STC-2 can act as a potent growth inhibitor and reduce intramembranous and endochondral bone development and skeletal muscle growth, implying that these tissues are specific physiological targets of stanniocalcins.stanniocalcins; stanniocalcin-related protein; development STANNIOCALCINS REPRESENT a small family of secreted homodimeric glycoprotein hormones consisting of STC-1 and STC-2, also known as stanniocalcin-related protein (STCrP), that has been conserved from aquatic to terrestrial vertebrates. STC-1 and STC-2 do not show significant homology to any other known proteins, and this has hampered understanding of their function(s). Initially, it was assumed that mammalian STC-1 would mimic the function of fish STC-1 in mineral homeostasis, and there is evidence to support this (26, 37, 52). Recently, however, it has become clear that STC-1 has a significantly expanded role in mammals on the basis of its expression pattern (7), gain-of-function transgenic mouse studies (13, 49), and subcellular localization (29, 38).STC-2 was initially identified as a stanniocalcin by virtue of its 50% identity and 73% amino acid homology to a stretch of 76 amino acids located between positions 24 and 101 of human (h)STC-1 (8, 12, 18, 32). hSTC-2 amino acid sequence downstream of position 101 shows less identity (23%) to hSTC-1, and it is 45 amino acids larger even though the genes encoding these proteins have identical intron/exon junctions (18). Unlike with STC-1, studies examini...

We have isolated a human cDNA clone encoding the mammalian homolog of stanniocalcin (STC), a calcium-and phosphate-regulating hormone that was first described in fishes where it functions in preventing hypercalcemia. STC has a unique amino acid sequence and, until now, has remained one of the few polypeptide hormones never described in higher vertebrates. Human STC (hSTC) was found to be 247 amino acids long and to share 73% amino acid sequence similarity with fish STC. Polyclonal antibodies to recombinant hSTC localized to a distinct cell type in the nephron tubule, suggesting kidney as a possible site of synthesis. Recombinant hSTC inhibited the gill transport of calcium when administered to fish and stimulated renal phosphate reabsorption in the rat. The evidence suggests that mammalian STC, like its piscine counterpart, is a regulator of mineral homeostasis.Stanniocalcin (STC) is a calcium-regulating hormone in bony fishes that has never been described in higher vertebrates, including mammals (1). The hormone is synthesized by the corpuscles of stannius (CS), endocrine glands that are associated with the kidneys of all fishes with a bony skeleton (2). The primary function of STC in fishes is the prevention of hypercalcemia and a rise in serum calcium levels is the primary stimulus for secretion (3). Upon release into the circulation, STC lowers calcium transport by the gills thereby reducing its rate of influx from the environment into the extracellular compartment (1). A second equally important action of STC is stimulation of phosphate reabsorption by renal proximal tubules (4). The consequence of this renal effect is increased levels of plasma phosphate, the latter of which combines with excess calcium and promotes its disposal into bone and scales. Because the CS have never been identified in higher vertebrates, it has long been assumed that STC was unique to fishes. However, recent evidence of STC immunoreactivity in human kidney and serum argues for a more widespread existence of the hormone (5).By a process of random sequencing of human tissue cDNAs, we have isolated a lung-derived cDNA clone whose deduced protein sequence bears a strikingly high level of homology to salmon and eel STC (6, 7). § Data indicating that human (h)STC inhibits calcium uptake in fish and phosphate excretion in rats suggest that hSTC is a hormonal regulator of mineral metabolism. MATERIALS AND METHODScDNA Isolation and Analysis. cDNA isolation. The initial expressed sequence tag (EST) clones used in the study were discovered by scientists at The Institute for Genomic Research by using established EST methods (8, 9). These clones wereThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.part of a larger EST project (10). This clone was used for rescreening the same library and a full-length clone encoding hSTC was obtained.Southern blot analysis. Ten-microgram aliquo...

Milk production is a function of the number and activity of mammary epithelial cells, regardless of stage of lactation. Milk yield is generally higher in multiparous cows than in primiparous cows, but persistency is usually greater in the latter group. We compared several measures related to metabolic activity, apoptosis, and endocrine control of mammary cell growth in 8 primiparous and 9 multiparous cows throughout lactation. Mammary gland biopsies were taken in early [10 d in milk (DIM)], peak (50 DIM), and late (250 DIM) lactation to evaluate gene expression and determine DNA and fatty acid synthase (FAS) content. Milk samples taken the day before the biopsies were used to detect protease activities and to determine stanniocalcin-1 (STC) concentrations. Blood samples served to measure insulin-like growth factor-1, prolactin, and STC concentrations. Milk yield was higher in multiparous cows than in primiparous cows at the 10 DIM (32.8 +/- 1.3 and 25.2 +/- 0.8 kg/d) and 50 DIM (38.0 +/- 1.2 and 29.8 +/- 1.1 kg/d), but it was the same for both groups at 250 DIM (23.9 +/- 1.5 and 23.8 +/- 1.1 kg/d). Except for stearoyl-coenzyme A desaturase, expression of genes related to milk synthesis was not affected by stage of lactation. However, gene expression of acetyl-coenzyme A carboxylase, beta-casein, and FAS was lower in early lactation in primiparous cows. Expression of both proapoptotic bax and antiapoptotic bcl-2 genes was higher in primiparous cows, whereas the bax-to-bcl-2 ratio was not changed. Mammary DNA concentration was higher in multiparous cows, as was the amount of FAS protein in early lactation. Two bands of protease activity were found in milk samples, and one of the bands had an apparent molecular weight similar to gelatinase A and was dependent on the stage of lactation. Serum insulin-like growth factor-1 increased with day of lactation and was higher in primiparous cows. Serum prolactin decreased in late lactation, but peak values were observed in early lactation for primiparous cows and peak lactation for multiparous cows. Milk STC content increased with advancing lactation. The results are consistent with a lower degree of differentiation and a greater capacity for cell renewal in the mammary gland of primiparous cows.