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Gabriel E. DiMattia, PhD Associate Professor Departments of Biochemistry and Oncology Our Research Program
The Molecular Endocrinology of Stanniocalcins (STC-1 & STC-2 or STCrP) A molecular genetic approach to determining their function and regulation in mammals What are STCs and why study them? 
STC-1 and -2 constitute a small family of homodimeric glycoprotein hormones conserved from fish to human. In fact, STC-1 was discovered in fish were it acts as a potent calcium-lowering hormone produced by a discrete endocrine organ, the corpuscles of Stannius, located on the fish kidney. The corpuscles of Stannius are not present in mammals, but STC-1 was discovered in mammals in 1995 (Proc Natl Acad Sci U S A. 1995 92(6):1871-5), and in 1996 several groups reported the isolation of the human STC-1 cDNA providing unequivocal proof of its existence in mammals. 
  

The identification of STC-1 in mammals was a significant advancement and naturally led many to speculate that STC-1 plays an important role in how calcium and phosphate levels are regulated in humans. This assumption was also fueled by the fact that the amino acid sequence of human STC-1 showed ~73% sequence similarity to fish STC-1. Consequently, it seemed reasonable and likely that this hormone was playing an important physiological role in mammals, but exactly what that role might be is open to investigation and could have important biomedical implications. Recently, we and others discovered a second stanniocalcin-like protein through mining the EST database, and have termed it STC related protein (STCrP) while others call it STC-2. Clearly, the potential clinical importance of STCs and the novelty of determining the function of newly identified regulatory factors were strong impetus to undertake a thorough study of STC biochemistry at the cellular level and in the whole animal. 
What do we know about STC-1 & -2? 
STC-1 and -2 do not show significant homology to any other known proteins and do not contain previously recognized protein motifs. 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 (Madsen et al., 1998; Olsen et al., 1996; Wagner et al., 1997). A number of studies have focused on where and when STC-1 is produced and make inferences regarding its function based on localization data, but relatively few studies have directly assessed the function of STC-1 (Chang et al., 2003). There is good evidence for a role for STC-1 in mammalian mineral metabolism. A bolus injection of recombinant hSTC-1 can significantly decrease renal phosphate excretion in a rat bioassay without changes in plasma electrolytes (Olsen et al., 1996; Wagner et al., 1997). Others have shown that recombinant hSTC-1 in swine duodenum preparations can decrease Ca2+ uptake and concomitantly increase PO4 reabsorption (Madsen et al., 1998). Our hSTC-1 gain-of-function transgenic mice showed significantly higher serum phosphate levels compared to age and sex-matched wild-type animals, but a significant change in serum Ca2+ was not observed across different transgenic lines and sexes (Varghese et al., 2002). Therefore, current data indicate that mammalian STC-1 can alter the movement of phosphate, but has little if any, direct effect on serum Ca2+ homeostasis. Interestingly, Zhang and co-workers (Zhang et al., 2000) showed that an increase in extracellular Ca2+ concentration increased STC1 gene expression by the Paju neuronal cell line and that phosphate uptake by these cells was significantly increased in cells treated with recombinant hSTC-1. Moreover, they showed that Paju cells transfected with an hSTC-1 expression vector resulted in significantly increased cell viability after treatment with cobalt chloride to induce hypoxia or thapsigargin to elevate intracellular Ca2+, both of which cause cell death. More recently, Sheikh-Hamad and coworkers (Sheikh-Hamad et al., 2003) reported that hSTC-1 treatment of rat cardiomyocytes in culture resulted in complete cessation of contraction after 25 minutes and this dramatic effect was correlated with a large reduction in Ca2+ current through L-channels indicating that STC-1 can inhibit L-channel activity. The aforementioned studies suggest that the action of STC-1 in regulating mammalian Ca2+ levels may lie primarily on intracellular pools rather than systemic Ca2+ regulation. With the advent of DNA microarray analysis of gene expression, induction of STC1 and STC2 expression has been correlated with a number of processes unrelated to mineral homeostasis DiMattia STC table2.htm. For instance, steady-state levels of STC-1 mRNA are increased in human umbilical vein endothelial cells treated with lysophosphatidylcholine, a proatherogenic factor [Sato et al.1998 J Biochem 123:1119-26]. Others have shown that treatment of these cells with stimulators of angiogenesis results in a 100-fold induction of STC-1 mRNA levels [Kahn et al. 2000 Am J Pathol 156:1887-900]. STC-1 mRNA levels also increase upon differentiation of human neural crest-derived cells (Paju line), suggesting that it may play a role in neuronal maturation or is induced as a consequence of neuronal development [Zhang et al. 1998 Am J Pathol 153:439-45]. Moreover, the STC1 gene is most highly expressed during mouse development in a variety of developing structures, intimating that it plays a role during embryogenesis. Recently, the subcellular localization of STC-1 has pointed to role(s) for it in the mitochondria and the nucleus (McCudden et al. 2002). Collectively, these observations seem to suggest that STC-1 may be involved in many physiological processes or may perform similar functions in different tissues. Considerably less is known regarding the function of STC-2 in mammals. STC-2 was discovered in 1998 through a nucleotide sequence homology search of the expressed sequence tag (EST) database by virtue of its similarity to STC-1 (Chang and Reddel, 1998; DiMattia et al., 1998; Ishibashi et al., 1998; Moore et al., 1999). It is tempting to assume that the function(s) of STC-1 and STC-2 overlap because of the similarity in primary amino acid sequence and conservation of key cysteine residues found in hSTC-1 (Moore et al., 1999). However there are distinct differences between these proteins, including the fact that STC-2 is ~20% larger, most of which is present in the form of a histidine rich COOH-terminal region absent in STC-1 (Moore et al., 1999). Moreover, the expression of STC-1 and -2 differ. The STC-1 expression in the mouse is ubiquitous, but it is most highly expressed during embryogenesis and in the adult ovary (Chang et al., 2003; Varghese et al., 1998). Our mouse STC-2 expression data indicates that unlike STC-1, STC-2 is not detectable in mouse embryo RNA by northern blotting, however, is detectable in a variety of adult tissues and fibroblasts isolated from mouse embryos (Gagliardi et al., 2004). Collectively these data suggest that the biological role(s) of STC-2 in mammals may differ from that of STC-1, and this is further supported by the fact that STC-2 is unable to displace STC-1 from its putative receptor in competition studies (Luo et al., 2004; McCudden et al., 2002). Studies directly assessing the function of STC-2, other than our recent report on human STC-2 transgenic mice (Gagliardi et al., 2004) have recently been reported. Gopal Thinakaran's lab reported that STC2 expression can be rapidly and strongly upregulated in a variety of cell types by endoplasmic reticulum stress which causes the unfolded protein response (Ito et al. 2004). Interestingly, STC1 expression was not similarily regulated by the unfolded protein response and unlike STC-1, STC-2 is not found in the mitochondria. Moreover, Ito and co-workers showed that overexpression of STC-2 conferred a cytoprotective effect on cells when challenged with ER stress modulators. Clearly, STC-2 functions in a paracrine manner in response to toxic insults and work of Ito et al. 2004 will be important in our interpretation of how STCs function in an overexpression model. STC hormones in cancer: It is clear that selective estrogen receptor modulators, such as tamoxifen, are effective in the treatment of many estrogen receptor-positive breast cancers because estrogen (E2) plays a critical role in the initiation and promotion of breast cancer (Gradishar and Jordan, 2003; Jordan, 2004). It is also important to recognize that the effects of E2 are mediated in large part by its nuclear localized receptor that acts as a transcription factor modulating the expression of a large complement of genes that dictate the estrogen response of a particular cell type. Consequently, there is great interest in identifying genes regulated by E2 in breast cancer because of their potential importance in the etiology of the disease and as potential downstream therapeutic targets. It is within this context that the stanniocalcin (STCs) hormones have received much attention recently. Serial Analysis of Gene Expression, or SAGE, indicated that STC-2 is most highly expressed in human mammary gland cells. SAGE is an experimental technique designed to gain a quantitative measure of gene expression and includes several steps utilizing molecular biological, DNA sequencing and bioinformatics techniques. SAGE established STC-2 as a secretory product of human breast cells implying that it plays a role in breast biology. Gene expression profiling studies, primarily with breast tumours or breast cancer cell lines treated with E2, as well as other tumour types have shown that either STC-1 or STC-2 expression is significantly altered in the tumour or E2 treated cells (Amatschek et al., 2004; Bouras et al., 2002; Charpentier et al., 2000; Frasor et al., 2003; Fujiwara et al., 2000; Garber et al., 2001; Gruvberger et al., 2001; Halmos et al., 2004; Ismail et al., 2000; Iwao et al., 2002; Iyengar et al., 2003; Lal et al., 2001; Liang et al., 1992; Liu et al., 2004; Planey et al., 2003; Porter et al., 2003; Schwartz et al., 2002; Wascher et al., 2003; Watanabe et al., 2002; Welcsh et al., 2002; Wilson et al., 2002). With regard to breast cancer, the majority of these reports have demonstrated that STC-2 expression is significantly upregulated in breast tumours (Amatschek et al., 2004; Gruvberger et al., 2001; Wilson et al., 2002), but down-regulation has also been reported (Planey et al., 2003; Porter et al., 2003). A number of reports have also shown that STC-2 expression is strongly elevated in E2-treated human breast cancer cell lines (Bouras et al., 2002; Charpentier et al., 2000; Frasor et al., 2003; Gruvberger et al., 2001). Moreover, mice in which the coactivator-associated arginine methyltransferase 1 (CARM1) gene has been knocked-out, exhibit a significant decrease in STC-2 expression because CARM1 is necessary for estrogen receptor (ER) activity (Yadav et al., 2003). More recently, Lin and co-workers (Lin et al., 2004) used the STC2 gene as an internal positive control for their search for ERalpha responsive elements and genes in breast cancer. Collectively, these reports strongly suggest that STC-2 is a direct target of activated ER in human breast cancer cells and that STC-2 plays a role in mediating the effects of estrogen on breast epithelial cells. 
Our research into the function of STC-1 & -2
Our approach to determining the function of STC hormones in mammals focusses on the use of the mouse as our model system. We began by isolating and characterizing the mouse and human STC1 genes and determining where STC-1 was expressed. Unexpectedly, our studies have shown that STC-1 is most highly expressed in the mouse ovary and specifically in theca-interstitial cells which are responsible for the production of androgens. The androgen is then converted to estrogen in the granulosa cells that surround the egg. STC-1 accumulates to high levels in the corpora lutea (progesterone-producing structure) and in the oocyte suggesting that STC-1 may be a newly discovered ovarian regulator. We've also discovered that the production of STC-1 in the ovary is significantly increased during pregnancy in the mouse and that this high level of expression also occurs during lactation. Interestingly, ovarian production of STC-1 during lactation is dependent upon the presence of a suckling litter. Therefore, it is tempting to speculate that STC-1 plays a role in preparing the mother for nursing. Taken together, these data imply that STC-1 functions as a regulator of the female reproductive system, not only in the ovary but perhaps, also in the mammary gland. Based on these data, we have focused our efforts on understanding what STC-1 is doing in the ovary and how its production is regulated there. However, our approach to this problem will inevitably shed new light on the function of STC-1 in all of the tissues that produce it and during development.  
Our approach to the problem 
We have chosen a molecular genetic approach to defining STC-1 & -2 function in the mouse model system. This typically involves gene knockout and overexpression in vitro or in vivo. These are complementary approaches that are often required because there are numerous reports (No OVERT phenoype Tgs.doc) ‬where overexpression of specific proteins lead to no obvious disturbance in animal physiology. Furthermore there are many reports were production of null alleles in mice do not produce an overt phenotype whereas overexpression of the protein (e.g., cytokine-inducible SH2‭ ‬domain-containing protein‭, ‬placenta growth factor‭)‬‭ ‬results in a distinct and informative phenotypic change. Given that there was no evidence for STC-1 function in mammals at the time, our laboratory chose the gain-of-function approach because of the relatively quick generation time and high efficiency of success in obtaining transgenic mice. Moreover, owing to independent chromosomal integration events, it is common to obtain lines of mice expressing different levels of the transgene thus providing a dose-response characteristic to the phenotype(s). The goal was to drive long-term, stable and ubiquitous transgene expression in developing embryos and adult mice thus providing constant exposure to the hormones. The expectation was that increased levels of STCs in circulation and constitutive exposure to STCs would lead to abnormal physiology in STC target tissues. Consequently, overt organ or system pathologies can result in areas of the body that are critically regulated by STCs and therein provide evidence of a physiological role for STCs in the tissues. This in turn would also allow further study of that tissue-type as a target of STCs and source of STC receptors. We have generated mice that constitutively produce human STC-1 or human STC-2, from early in development. Interestingly, hyperstimulation by these hormones results in early embryonic growth restriction which continues post-natally such that mature mice are, on average, 45% smaller than their non-transgenic litteremates. We have also shown that STC-2 overexpression leads to neonatal death in a proportion of newborn pups indicating that hyperstimulation by STC-2 can have a debilitating effect on neonatal physiology. The dramatic reduction in growth may be linked to the effects of these hormones on bone development because we have shown that skull bone growth is significantly delayed (see below) and that long bone ossification is also hampered in the embryo. Consequently, one of the major functions of STC hormones may be in the development and growth of the skeleton. This is further supported by the work of others, showing that STC-1 can enhance osteoblast differentiation in culture (Yoshiko et al., 2003) and that the highest levels of embryonic Stc1 gene expression are in the intervertebral discs and developing joints (Stasko and Wagner, 2001; Yoshiko et al., 2002). Recent analysis of Stc2 expression in the avian embryo shows that it is highly expressed in the developing joints similar to Stc1. Our transgenic mice have also revealed that ectopic production of human STC-1 severely compromises female reproductive ability whereas male reproductive potential is not significantly altered. In fact, the testes of STC-2 transgenic mice are twice the expected size given the reduced weight of the animals. It is also notable that the reduction in size (both weight and length) of the transgenic mice is not dependent upon a reduction in growth hormone (GH) and insulin-like growth factors (IGF). These are the primary factors controlling body growth and mouse mutants that exhibit growth reduction are often linked to changes in GH and IGFs. We believe that understanding the mechanism by which STCs inhibit growth will reveal their function in mammals. Consequently, our studies, at the molecular level, are focussed on how STC hormones are changing cell physiology and we are utilizing a variety of techniques to attack this biochemical problem. Technologies utilized 
Our research is primarily based in molecular biology with an emphasis on examining the expression and structures of genes in mice and humans. This work not only involves primary tissues but also makes use of different immortalized cell lines for transcriptional studies. We also use a variety of biological systems to produce recombinant proteins for in vitro and in vivo studies. Histology is a key technique for the analysis of novel mouse phenotypes produced by genetic manipulation. Significance to human disease
Our research has clearly shown that STC-1 and STC-2 are potent regulatory molecules in the whole animal. As stated above and listed in the embedded table DiMattia STC table2.htm, the expression of STC hormones appears to be linked to a variety of disease states including cancer. In fact, it is clear from gene profiling studies that these hormones are highly responsive to a number of stressors including viral infection, DNA damaging agents, and carcinogens. Consequently, the available evidence strongly suggest that STCs are important physiological regulators and identifying their cellular and molecular targets will be key to determining which disease states are particularly affected by their action. It is intriguing that STC-2 is highly expressed in human breast cancer and that STC-1 and -2 can affect bone physiology given that the skeleton is the preferred target of metastatic human breast cancer cells. Bone metastases are indeed found in virtually all advanced breast cancer patients. Therefore, like osteopontin, the expression of STC-2 in breast cancer may play a role in the spread of breast cancer cells to the bone. This potential relationship is clinically relevant and clearly warrants further investigation.http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7892193&dopt=Abstractresearch program_files/DiMattia%20STC%20table2.htmhttp://mcb.asm.org/cgi/content/abstract/24/21/9456?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=gopal&searchid=1097854288062_1965&stored_search=&FIRSTINDEX=0&volume=24&issue=21&journalcode=mcbhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14871811http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11888893http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11085516http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12959972http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10717250http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11707590http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11507038http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15205324http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11118061http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11809729http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14508521http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11535709http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=1458489http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14996724http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12517795http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12651909http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12183431http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12684415http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12107109http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12032322http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12368191http://www.biochem.uwo.ca/fac/dimattia/Specific%20protein%20methylation%20defects%20and%20gene%20expression%20perturbationshttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15345050http://www.ensembl.org/Mus_musculus/protview?peptide=ENSMUSP00000020546research program_files/No%20OVERT%20phenoype%20Tgs-1.dochttp://mcb.asm.org/cgi/content/full/19/9/6396?view=long&pmid=10454585http://ajrccm.atsjournals.org/cgi/content/full/169/4/505http://www.springerlink.com/(wu40ocj00btq31yieaipam55)/app/home/contribution.asp?referrer=parent&backto=issue,12,22;journal,1,758;linkingpublicationresults,1:100395,1research program_files/DiMattia%20STC%20table2_1.htmshapeimage_5_link_0shapeimage_5_link_1shapeimage_5_link_2shapeimage_5_link_3shapeimage_5_link_4shapeimage_5_link_5shapeimage_5_link_6shapeimage_5_link_7shapeimage_5_link_8shapeimage_5_link_9shapeimage_5_link_10shapeimage_5_link_11shapeimage_5_link_12shapeimage_5_link_13shapeimage_5_link_14shapeimage_5_link_15shapeimage_5_link_16shapeimage_5_link_17shapeimage_5_link_18shapeimage_5_link_19shapeimage_5_link_20shapeimage_5_link_21shapeimage_5_link_22shapeimage_5_link_23shapeimage_5_link_24shapeimage_5_link_25shapeimage_5_link_26shapeimage_5_link_27shapeimage_5_link_28shapeimage_5_link_29shapeimage_5_link_30shapeimage_5_link_31
Perhaps grooming a little too energetically? Can you identify the STC overexpressing transgenic pups in this litter? Publications Links Home Contact Information Yudith Ramos-Valdes Research Technician Early STC-2 induced embryonic growth restriction Developmental delay in cranial flat bone development caused by exposure to STCs
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