Biomedical subjects

K W Sloop

Publications and source records attributed to K W Sloop.

7 recordsLinked to original sources

PubMed2000

Biochemical and genetic characterization of the porcine Prophet of Pit-1 pituitary transcription factor.

Prophet of Pit-1 (Prop-1) is a paired class homeodomain transcription factor that is specifically expressed in the pituitary gland. Mutations in the Prop-1 gene cause compound pituitary diseases in mouse models and human patients. We have cloned and analyzed the porcine ortholog of Prop-1. Analysis of cDNAs revealed that the porcine Prop-1 sequence is similar to the mouse and human proteins within the homeodomain and carboxyl terminus, but the amino terminus is poorly conserved. The Prop-1 gene consists of three exons and two introns and spans 3.8 kilobases of genomic DNA. In addition, we mapped Prop-1 to the q arm of pig chromosome two. During development, Prop-1 is expressed at the time of pituitary organogenesis. In the adult, expression was observed at low levels only in the pituitary gland. The porcine Prop-1 protein displays similar biochemical, DNA binding, and transcriptional activities to human PROP-1. We conclude that, although the structural divergence between the porcine and human PROP-1 molecules may indicate some distinct functions, the porcine Prop-1 gene encodes a pituitary transcription factor with similar overall activities to the human ortholog.

Amino Acid Sequence↗

PubMed2000

Analysis of the human LHX3 neuroendocrine transcription factor gene and mapping to the subtelomeric region of chromosome 9.

The Lhx3 LIM homeodomain transcription factor is critical to pituitary organogenesis and motor neuron development. We determined the genomic structure and chromosomal localization of human LHX3. The gene contains seven coding exons and six introns that span 8.7 kilobases in length. The LHX3 gene codes for two functionally distinct isoforms that differ in their amino termini but share common LIM domains and a homeodomain. The functional domains of the LHX3 proteins are encoded by distinct exons. The alternate amino termini and LIM domains lie within individual exons, and the homeodomain is coded by two exons interrupted by a small intron. Human LHX3 maps to the subtelomeric region of chromosome 9 at band 9q34.3, within a region noted for chromosomal translocation and insertion events. Characterization of the genomic organization and chromosomal localization of LHX3 will enable molecular evaluation and genetic diagnoses of pituitary diseases and central nervous system developmental disorders in humans.

Amino Acid Sequence↗

PubMed2000

Molecular analysis of LHX3 and PROP-1 in pituitary hormone deficiency patients with posterior pituitary ectopia.

The cause of posterior pituitary ectopia associated with anterior pituitary hormone deficiencies is unknown. We describe children with combined pituitary hormone deficiency (CPHD) or isolated GH deficiency. In all cases, magnetic resonance imaging examination revealed abnormal pituitary gland development featuring ectopic posterior lobe location and frequently hypoplastic anterior lobes. Embryonic development of the pituitary requires the coordinated expression of specific transcription factors. Mutations of the PIT-1 and PROP-1 transcription factors are responsible for CPHD in some patients with normally positioned posterior pituitaries. In mice, the Lhx3 LIM homeodomain transcription factor is required for both structural development and cellular differentiation of the pituitary gland. Thus, we hypothesized that mutations in one or both of the two human LHX3 isoforms are responsible for posterior pituitary ectopia associated with anterior pituitary hypopituitarism. Comprehensive molecular analysis of the LHX3 isoforms was performed to test this hypothesis. No loss of function mutations in the LHX3 gene were detected. In addition, analysis of PROP-1 did not reveal mutations that might cause this phenotype. These studies suggest that the abnormal processes leading to the development of CPHD or GH deficiency associated with posterior pituitary ectopia are not a result of aberrant LHX3 or PROP- 1 function, but may be caused by defects at other gene loci.

Animals↗

PubMed1999

Differential activation of pituitary hormone genes by human Lhx3 isoforms with distinct DNA binding properties.

Lhx3 is a LIM homeodomain transcription factor essential for pituitary development and motor neuron specification in mice. We identified two isoforms of human Lhx3, hLhx3a and hLhx3b, which differ in their ability to trans-activate pituitary gene targets. These factors are identical within the LIM domains and the homeodomain, but differ in their amino-terminal sequences preceding the LIM motifs. Both isoforms are localized to the nucleus and are expressed in the adult human pituitary, but gene activation studies demonstrate characteristic functional differences. Human Lhx3a trans-activated the alpha-glycoprotein subunit promoter and a reporter construct containing a high-affinity Lhx3 binding site more effectively than the hLhx3b isoform. In addition, hLhx3a synergized with the pituitary POU domain factor, Pit-1, to strongly induce transcription of the TSHbeta-subunit gene, while hLhx3b did not. We demonstrate that the differences in gene activation properties between hLhx3a and hLhx3b correlate with their DNA binding to sites within these genes. The short hLhx3b-specific amino-terminal domain inhibits DNA binding and gene activation functions of the molecule. These data suggest that isoforms of Lhx3 may play distinct roles during development of the mammalian pituitary gland and other neuroendocrine systems.

Amino Acid Sequence↗

PubMed1998

Changes in leptin expression are not associated with corresponding changes in CCAAT/enhancer binding protein-alpha.

C/EBP-alpha binds a C/EBP consensus site in the leptin promoter and activates transcription in vitro. We assessed adipose tissue expression of C/EBP-alpha, leptin and beta-actin in Sprague Dawley rats under conditions that modulate leptin mRNA abundance in order to study the relationship between leptin and C/EBP-alpha expression patterns. During acute fasting, which decreased the level of leptin and beta-actin mRNA, C/EBP-alpha mRNA expression was unaltered. In leptin-treated and pair-fed animals, C/EBP-alpha mRNA was unaltered compared to ad libitum fed controls, while leptin and beta-actin mRNA expression was again decreased. These results indicate that changes in the level of leptin gene expression are not directly associated with changes in the level of C/EBP-alpha abundance.

Adipose Tissue↗

PubMed1998

Differentiation method-dependent expression of leptin in adipocyte cell lines.

Leptin, the product of the ob gene, is expressed exclusively in adipose tissue. However, adipocyte cell lines, such as 3T3-L1 adipocytes, have generally been reported to express extremely low levels of leptin mRNA. We compared 3T3-L1's to the closely related line 3T3-F442A, and to another murine adipocyte line, TA1. TA1 cells, when differentiated by indomethacin/insulin treatment, express leptin at levels greater than those of 3T3-L1 adipocytes differentiated by the traditional methylisobutylxanthine/dexamethasone/insulin protocol. However, when 3T3-L1's are differentiated in the presence of indomethacin/insulin their expression levels of leptin increase dramatically. 3T3-F442A preadipocytes also express high levels of leptin when differentiated in the presence of T3 and insulin, but when differentiated in the presence of indomethacin/insulin, expression levels drop precipitously. These changes in leptin mRNA and protein expression are not reflected by changes in CCAAT/enhancer binding protein-alpha (c/EBPalpha), peroxizomal proliferator activated receptor-gamma (PPARgamma), lipoprotein lipase (LPL), fatty-acid binding protein aP2 or uncoupling protein-2 (UCP2) mRNA levels, and suggest a mechanism unique to the leptin gene.

3T3 Cells↗

PubMed1996

Regulation of expression of ob mRNA and protein by glucocorticoids and cAMP.

Regulation of obese gene (ob) expression in ob/ob and db/db mice and in cultured rat adipocytes was examined. It has been demonstrated that exogenous human OB protein (leptin) treatment reduces food intake and weight gain, as well as insulin, glucose, and corticosterone levels in ob/ob mice. In the present report we show that leptin treatment down-regulates endogenous adipose ob mRNA. However, treatment of isolated rat adipocytes with 100 ng/ml human or murine leptin had no direct effect on expression of endogenous ob mRNA, suggesting that leptin may be able to down-regulate its own expression by an indirect, non-autocrine mechanism. Glucocorticoids increased both ob mRNA levels and secreted leptin levels in vitro. Conversely, agents that increase intracellular cAMP, such as beta-adrenergic agonists or Bt2cAMP itself, decreased ob mRNA expression and leptin secretion. Therefore, increased glucocorticoid levels and decreased sympathetic neural activity may contribute to the elevated ob mRNA expression observed in genetically obese, hyperglucocorticoid rodents. Furthermore, leptin might regulate its own expression through a feedback mechanism involving the hypothalamic pituitary axis.

Adipocytes↗