Margaret E. Bock

Differential binding of rat pituitary-specific nuclear factors to the 5′-flanking region of pituitary and placental members of the human growth hormone gene family

SummaryPlacental chorionic somatomammotropin (hCS-A or B) and growth hormone variant (hGH-V) are members of the human growth hormone family, and are related by structure and function to pituitary growth hormone (hGH-N). However, while the hGH-N gene is expressed specifically in the anterior pituitary, hGH-V and hCS are produced in the placenta. Hybrid hGH-N, hGH-V and hCS-A genes containing 5′-flanking sequences, including the endogenous promoter, are preferentially expressed in rat pituitary tumor (GC) cells, after gene transfer. Since interaction with a pituitary-specific protein (Pit 1) is required for efficient hGH-N as well as rat growth hormone (rGH) gene expression in GC cells, binding of pituitary proteins to the hGH-V and hCS-A promoter sequences was investigated. Rat Pit 1 binds at two locations on the hGH-N gene, a distal (−140/−107) and proximal site (−97/−66), in a similar manner to that observed with the rGH gene. By contrast, efficient Pit 1 binding was seen only to the distal site of the hGH-V gene and the proximal site of the hCS-A gene. Although binding of a protein to the distal hCS-A sequences was observed, the site of interaction was truncated (−140/−116), not pituitary-specific, and was more consistent with the binding of Sp1. These data indicate that rat Pit 1 binds to the placental hGH-V and hCS-A genes and correlates with their promoter activity in GC cells after gene transfer. However, the data also indicate that rat Pit 1 binds to human and rat pituitary growth hormone in a similar manner (two sites of interaction) and that the pattern of binding is distinct from the placental members of the hGH gene family. These data indicate that human Pit 1, unlike the rat equivalent, might distinguish these genes functionally (tissue-specifically) as well as structurally.

Detection of placental growth hormone variant and chorionic somatomammotropin-L RNA expression in normal and diabetic pregnancy by reverse transcriptase-polymerase chain reaction

Diabetes is a common complication encountered during pregnancy. Earlier studies indicated that diabetic placentas bear morphological alterations consistent with modified placental differentiation, including alterations in the villous cellular content, structure, and total surface. Limited data associating the diabetic status with the expression of terminal placental differentiation markers are available. The human growth hormone/chorionic somatomammotropin (hGH/CS) family consists of five genes, one of which (GH-N) is expressed efficiently in pituitary while the other four (CS-A, B, L, and hGH-V) are expressed in placenta and represent ultimate placental differentiation markers. We developed and applied a sensitive RT-PCR method coupled with diagnostic restriction digestion to determine the relative levels of the hGH/CS family in normal pregnancies and examine whether their mRNA expression pattern is altered in pregnancies complicated by diabetes. We show that relative hCS-L content changes during placental development. Specifically, normal term placentas express higher relative levels of hCS-L, lower relative hGH-V levels and a 70-fold lower hGH-V/CS-L mRNA ratio compared to early placentas. Also, many term placentas from diabetic pregnancies express lower relative levels of hCS-L mRNA and a much higher hGH-V/CS-L mRNA ratio compared to normal term placenta, resembling more an early placenta pattern of expression. Thus, our study suggests that the expression of terminal placental differentiation markers, such as the hGH/CS genes, is altered in term placentas from these diabetics reflecting either impaired placental differentiation or post-differentiation impairment of normal placental function.

Embryonic Survival and Severity of Cardiac and Craniofacial Defects Are Affected by Genetic Background in Fibroblast Growth Factor-16 Null Mice

Disruption of the X-chromosome fibroblast growth factor 16 (Fgf-16) gene, a member of the FGF-9 subfamily with FGF-20, was linked with an effect on cardiac development in two independent studies. However, poor trabeculation with lethality by embryonic day (E) 11.5 was associated with only one, involving maintenance in Black Swiss (Bsw) versus C57BL/6 mice. The aim of this study was to examine the potential influence of genetic background through breeding the null mutation onto an alternate (C57BL/6) background. After three generations, 25% of Fgf-16(-/Y) mice survived to adulthood, which could be reversed by reducing the contribution of the C57BL/6 genetic background by back crossing to another strain. There was no significant difference between FGF-9 and FGF-20 RNA levels in Fgf-16 null versus wild-type mice regardless of strain. However, FGF-8 RNA levels were reduced significantly in Bsw but not C57BL/6 mice. FGF-8 is linked to anterior heart development and like the FGF-9 subfamily is reportedly expressed at E10.5. Like FGF-16, neuregulin as well as signaling via ErbB2 and ErbB4 receptors have been linked to trabeculae formation and cardiac development around E10.5. Basal neuregulin, ErbB2, and ErbB4 as well as FGF-8, FGF-9, and FGF-16 RNA levels varied in Bsw versus C57BL/6 mice. These data are consistent with the ability of genetic background to modify the phenotype and affect embryonic survival in Fgf-16 null mice.

Differential expression of human placental growth hormone variant and chorionic somatomammotropin genes in choriocarcinoma cells treated with methotrexate

Chorionic somatomammotropin (hCS) genes (hCS-A and hCS-B) and the placental growth hormone variant (hGH-V) gene are expressed in the syncytiotrophoblast in vivo, and at low levels in cytotrophoblast-like choriocarcinoma (BeWo) cells. Treatment of choriocarcinoma cells with methotrexate (MTX) will induce a cell type intermediate between a cytotrophoblast and syncytiotrophoblast. After treatment with MTX, hCS/hGH-V mRNA levels were decreased in BeWo cells, and only hGH-V and minor hCS-A related transcripts of 1.6, 2.1 and 4.2 kilobases, termed hCS-A2, hCS-A3 and hCS-A4, respectively, were detected. By contrast, chorionic gonadotropin RNA levels were increased. This pattern of hCS/hGH-V expression resembles that observed when BeWo cells are grown in thyroid hormone (T3)-depleted serum, where hGH-V/hCS RNA increases in response to T3. This increase is blunted by MTX treatment, but is not due to a decrease in number or affinity of T3 receptors. These data indicate that the hGH-V and hCS genes can be differentially regulated by MTX, and are consistent with MTX interfering with T3 responsiveness of these genes. Also, if BeWo cells treated with MTX do represent a transitional state, these data raise the possibility that hGH-V and hCS possess a different temporal pattern of expression in the developing trophoblast.

Chorionic gonadotrophin and c-myc expression in growing and growth-inhibited (intermediate) trophoblasts.

FEG-3 cells are a clonal line of human choriocarcinoma and resemble villous cytotrophoblasts which are the stem cells for the syncytiotrophoblast in the placenta. FEG-3 cells synthesize and secrete the alpha subunit of human chorionic gonadotrophin (hCG). Treatment of FEG-3 cells with the chemotherapeutic drug (1 microM) methotrexate (MTX) results in an increase in nuclear diameter. Cell division is blocked and a decrease in c-myc mRNA levels in observed. The effects on cell growth and c-myc mRNA expression are reversible, and cells treated with MTX for 48 h retain their proliferative potential. Assessment of placental hormone gene expression reveals that a member of the human growth hormone gene family is expressed at extremely low levels and is unaffected by MTX treatment. Alpha and beta chorionic gonadotrophin (hCG) levels are increased by MTX treatment, but levels decrease following removal of MTX. In contrast to hCG in FEG-3 cells, non-trophoblastic or ectopic production of alpha hCG in human cervical carcinoma cells is inhibited by MTX treatment. These data indicate that MTX will induce morphological and biochemical changes in FEG-3 cells. They reveal an inverse relationship between c-myc and hCG RNA expression, and suggest different mechanisms govern trophoblast versus non-trophoblast production of alpha hCG.

Characterization of fibroblast growth factor receptor 1 RNA expression in the embryonic mouse heart.

We used reverse transcriptase-polymerase chain reaction (RT-PCR) to clone fibroblast growth factor receptor (FGFR) 1 isoforms from embryonic mouse heart and as a more sensitive method to characterize FGFR1 RNA expression in embryonic and adult mouse hearts. We describe the cloning of both full-length short (2259 base pairs) and long (2526 base pairs) FGFR1 isoform cDNAs which generated 86 and 102 kilodalton proteins, respectively, following in vitro translation. An assessment of FGFR1 RNA indicates that FGFR1-IIIc is the major form in both the embryonic and adult heart but there is an approximately 8.5-fold decrease in RNA levels in the adult. Differential RNA blotting as well as RT-PCR analyses are consistent with a switch in the relative expression of the short versus long FGFR1 isoforms during heart development. The long isoforms are more abundant in the embryo and the short isoforms predominate in the adult. This may be important in the regulation of growth and development of the heart.

A useful model to compare human and mouse growth hormone gene chromosomal structure, expression and regulation, and immune tolerance of human growth hormone analogues

Human (h) pituitary growth hormone (GH) is both physiologically and clinically important. GH reaches its highest circulatory levels in puberty, where it contributes to energy homeostasis and somatogenic growth. GH also helps to maintain tissues and organs and, thus, health and homeostasis. A reduction in the rate of hGH production begins in middle age but if GH insufficiency occurs this may result in tissue degenerative and metabolic diseases. As a consequence, hGH is prescribed under conditions of GH deficiency and, because of its lipolytic activity, stimulation of hGH release has also been used to treat obesity. However, studies of normal GH production and particularly synthesis versus secretion are not feasible in humans as they require sampling normal pituitaries from living subjects. Furthermore, human (or primate) GH structure and, as such, regulation and potential function, is distinct from non-primate rodent GH. As a result, most information about hGH regulation comes from measurements of secreted levels of GH in humans. Thus, partially humanized hGH transgenic mice, generated containing fragments of human chromosome 17 that include the intact hGH gene locus and many thousands of flanking base pairs as well as the endogenous mouse (m) GH gene provide a potentially useful model. Here we review this mouse model in terms of its ability to allow comparison of hGH versus mGH gene expression, and specifically: (i) GH locus structure as well as regulated and rhythmic expression; (ii) their ability to model a clinical assessment of hGH production in response to overeating and hyperinsulinemia as well as a possible effect of exercise, and (iii) their hGH-related immune tolerance and thus potential for testing hGH-related analogue immunogenicity.

Expression of Placental Members of the Human Growth Hormone Gene Family Is Increased in Response to Sequential Inhibition of DNA Methylation and Histone Deacetylation

Abstract The genes coding for human (h) chorionic somatomammotropin (CS), hCS-A and hCS-B, and placental growth hormone (GH-V), hGH-V, are located at a single locus on chromosome 17. Efficient expression of these placental genes has been linked to local regulatory (5′ P and 3′ enhancer) sequences and a remote locus control region (LCR), in part, through gene transfer in placental and nonplacental tumor cells. However, low levels of endogenous hCS/GH-V transcripts are reported in the same cells compared with term placenta, suggesting that chromatin structure, or regulatory region accessibility, versus transcription factor availability contributes to the relatively low levels. To assess individual hCS-A, CS-B, and GH-V gene expression in placental and nonplacental tumor cells and the effect of increasing chromatin accessibility by inhibiting DNA methylation and histone deacetylation using 5-aza-2′-deoxycytidine (azadC) and trichostatin A (TSA). Low levels of hCS-A, CS-B, and GH-V were detected in placental and nonplacental tumor cells compared with term placenta. A significant >5-fold increase in activity was seen in placental, but not nonplacental, cells transfected with hybrid hCS promoter luciferase genes containing 3′ enhancer sequences. Pretreatment of placental JEG-3 cells with azadC resulted in a >10-fold increase in hCS-A, CS-B, and GH-V RNA levels with TSA treatment compared with TSA treatment alone. This effect was specific as reversing the treatment regimen did not have the same effect. An assessment of hyperacetylated H3/H4 in JEG-3 cells treated with azadC and TSA versus TSA alone revealed significant increases consistent with a more open chromatin structure, including the hCS 3′ enhancer sequences and LCR. These observations suggest that accessibility of remote and local regulatory regions required for efficient placental hGH/CS expression can be restricted by DNA methylation and histone acetylation status. This includes restricting access of the hCS 3′ enhancer sequences to available placental enhancer transcription factors.