Expression, Purification, and Bioassay of Human Stanniocalcin from Baculovirus-Infected Insect Cells and Recombinant CHO Cells

Zhang, Junli; Alfonso, Pedro J.; Thotakura, N. Rao; Su, Jeffrey; Buergin, Markus; Parmelee, David C.; Collins, A.; Oelkuct, M; Gaffney, Stacie; Gentz, Solange; Radman, Dennis P.; Wagner, Graham F.; Gentz, Reiner

doi:10.1006/prep.1997.0857

Cited by 35 publications

(24 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Blood was allowed to coagulate at room temperature, and serum was collected as the supernatant from two consecutive 18°C 20-min centrifugations at 15,000 g. Mouse serum hSTC-2 was characterized by separating 3 l of a 1:10 dilution or 1 l of serum proteins by SDS-PAGE in 12% gels. These gels were Western blotted with the use of a 1:5,000 dilution of rabbit anti-hSTC-2 antiserum raised by Veterinary Services, University of Western Ontario, against recombinant hSTC-2 provided by Human Genome Sciences (Rockville, MD) (60). The apparent molecular weight of transgene-derived hSTC-2 was compared with hSTC-2 produced by the MCF-7 human breast carcinoma cell line by Western blotting.…”

Section: Methodsmentioning

confidence: 99%

Human stanniocalcin-2 exhibits potent growth-suppressive properties in transgenic mice independently of growth hormone and IGFs

Gagliardi¹,

Kuo²,

Raulic³

et al. 2005

American Journal of Physiology-Endocrinology and Metabolism

Self Cite

111

114

View full text Add to dashboard Cite

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...

show abstract

Section: Methodsmentioning

confidence: 99%

Human stanniocalcin-2 exhibits potent growth-suppressive properties in transgenic mice independently of growth hormone and IGFs

Gagliardi¹,

Kuo²,

Raulic³

et al. 2005

American Journal of Physiology-Endocrinology and Metabolism

Self Cite

111

114

View full text Add to dashboard Cite

show abstract

“…20 STC1 protein was expressed in a baculovirus expression system and is greater than 90% pure. 21 Endotoxin levels of the STC1 preparation are not detectable. 21 Detail information about materials and methods is provided in the supplemental materials (available online at http://atvb.ahajournals.org).…”

Section: Chemicals and Reagentsmentioning

confidence: 99%

Human Stanniocalcin-1 Blocks TNF-α–Induced Monolayer Permeability in Human Coronary Artery Endothelial Cells

Chen

Jamaluddin

Yan

et al. 2008

ATVB

View full text Add to dashboard Cite

Objective-Our previous studies revealed upregulation of stanniocalcin-1 (STC1) in cardiac vessels in dilated cardiomyopathy. However, the functional significance of STC1 is unknown. The objective of this study was to determine the effects of STC1 on TNF-␣-induced monolayer permeability of human coronary artery endothelial cells (HCAECs). Methods and Results-Cells were pretreated with STC1 for 30 minutes followed by treatment with TNF-␣ (2 ng/mL) for 24 hours. Monolayer permeability was studied using a transwell system. STC1 pretreatment significantly blocked TNF-␣-induced monolayer permeability in a concentration-and time-dependent manner. STC1 effectively blocked TNF-␣-induced downregulation of endothelial tight junction proteins zonula occluden-1 and claudin-1 at both mRNA and protein levels. STC1 also significantly decreased TNF-␣-induced superoxide anion production. The inhibitory effect of STC1 was specific to TNF-␣, as it failed to inhibit VEGF-induced endothelial permeability. Key Words: stanniocalcin-1 Ⅲ TNF-␣ Ⅲ endothelial cell Ⅲ permeability Ⅲ superoxide anion S tanniocalcin 1 (STC1) is a 25-kDa disulfide-linked homodimeric glycoprotein, which was originally identified as a secretory hormone of the corpuscles of Stannius, an endocrine gland unique to bony fish. 1 The mammalian homolog of STC1 has been found in many species including humans, rats, and mice. 2,3 Human STC1 is encoded by a single copy gene located on chromosome 8p11.2-p21 and comprises 4 exons that encode 247 amino acids with 11 cysteine residues. 2,3 Human and mouse STC1 proteins have 98% amino acid identity and share 73% overall sequence homology to fish STC. 2 Unlike the localized expression of STC1 in fish, mammalian STC1 is not detected in the blood and is expressed in many organs including the kidney, intestine, heart, thyroid, lung, placenta, brain, and bone, 4,5 suggesting a paracrine rather than an endocrine role for STC1 in mammals. The primary function of STC in fish is to prevent hypercalcemia 6 by inhibiting calcium influx through the gills and gut and stimulating phosphate reabsorption by the kidneys. 7 Similarly, in rodents, STC1 reduces the intestinal uptake of calcium while enhancing that of phosphate. 8 Furthermore, this gene seems to play diverse roles in numerous developmental, physiological, and pathological processes including cancer, pregnancy, lactation, angiogenesis, organogenesis, cerebral ischemia, and hypertonic stress. 9 Studies also indicate that STC1 modulates inflammation. For example, STC1 inhibited chemotaxis of mouse macrophages and human monoblasts in response to monocyte chemotactic protein-1 (MCP-1) and stromal cell-derived factor-1 alpha (SDF1-␣) 10 and decreased transmigration of macrophages and T-lymphocytes across quiescent or interleukin (IL)-1 beta (IL-1␤)-activated human endothelial cells. 11 Despite the growing body of knowledge about STC1, little is known about its effects on the vascular system and how it might affect endothelial barrier function during inflammation.The endothelial barrier i...

show abstract

“…STC1-specific rabbit polyclonal antibodies (that do not cross react with STC2) and recombinant hSTC1 protein were kindly provided by Dr. Henrik Olsen, Human Genome Sciences (Rockville, MD). Recombinant human STC1 protein was expressed in a baculovirus expression system and is Ͼ90% pure (6,27).…”

Section: Patientsmentioning

confidence: 99%

Stanniocalcin-1 is a naturally occurring L-channel inhibitor in cardiomyocytes: relevance to human heart failure

Sheikh-Hamad

Bick

et al. 2003

American Journal of Physiology-Heart and Circulatory Physiology

View full text Add to dashboard Cite

Cardiomyocytes of the failing heart undergo profound phenotypic and structural changes that are accompanied by variations in the genetic program and profile of calcium homeostatic proteins. The underlying mechanisms for these changes remain unclear. Because the mammalian counterpart of the fish calcium-regulating hormone stanniocalcin-1 (STC1) is expressed in the heart, we reasoned that STC1 might play a role in the adaptive-maladaptive processes that lead to the heart failure phenotype. We examined the expression and localization of STC1 in cardiac tissue of patients with advanced heart failure before and after mechanical unloading using a left ventricular assist device (LVAD), and we compared the results with those of normal heart tissue. STC1 protein is markedly upregulated in cardiomyocytes and arterial walls of failing hearts pre-LVAD and is strikingly reduced after LVAD treatment. STC1 is diffusely expressed in cardiomyocytes, although nuclear predominance is apparent. Addition of recombinant STC1 to the medium of cultured rat cardiomyocytes slows their endogenous beating rate and diminishes the rise in intracellular calcium with each contraction. Furthermore, using whole cell patch-clamp studies in cultured rat cardiomyocytes, we find that addition of STC1 to the bath causes reversible inhibition of transmembrane calcium currents through L-channels. Our data suggest differential regulation of myocardial STC1 protein expression in heart failure. In addition, STC1 may regulate calcium currents in cardiomyocytes and may contribute to the alterations in calcium homeostasis of the failing heart.

show abstract

Expression, Purification, and Bioassay of Human Stanniocalcin from Baculovirus-Infected Insect Cells and Recombinant CHO Cells

Cited by 35 publications

References 23 publications

Human stanniocalcin-2 exhibits potent growth-suppressive properties in transgenic mice independently of growth hormone and IGFs

Human stanniocalcin-2 exhibits potent growth-suppressive properties in transgenic mice independently of growth hormone and IGFs

Human Stanniocalcin-1 Blocks TNF-α–Induced Monolayer Permeability in Human Coronary Artery Endothelial Cells

Stanniocalcin-1 is a naturally occurring L-channel inhibitor in cardiomyocytes: relevance to human heart failure

Contact Info

Product

Resources

About