Biomedical subjects

James M Hammond

Publications and source records attributed to James M Hammond.

5 recordsLinked to original sources

PubMed2005

Follicle-stimulating hormone induction of ovarian insulin-like growth factor-binding protein-3 transcription requires a TATA box-binding protein and the protein kinase A and phosphatidylinositol-3 kinase pathways.

The current study was done to elucidate the mechanism of the FSH stimulation of IGF-binding protein 3 (IGFBP-3) expression and map the FSH response element on the pig IGFBP-3 promoter. Forskolin induced IGFBP-3 reporter activity in transiently transfected granulosa cells. The protein kinase A (PKA) inhibitor [N-[2-(p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2HCl] (and cotransfection with a PKA inhibitor expression vector), the phosphatidylinositol-3 kinase inhibitor [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], and the ERK inhibitor [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene], all blocked FSH stimulation. Use of serial deletion constructs and site-directed mutagenesis show that a TATA box-binding protein site is required for FSH stimulation and that a specific protein 1 (Sp1) site is required for basal transcription. Gel shift assays of nuclear protein with a -61/-25 probe detected four protein-DNA complexes, with bands I and II having significantly higher intensities in FSH-treated cells than in controls. Mutation of the Sp1 site prevented formation of bands I and II whereas mutation of the TATA box-binding protein site prevented formation of band IV. Use of specific antibodies showed that Sp1 participates in formation of band I, Sp3 band II, and p300 in both I and II. Band III was nonspecifically competed out. We conclude that FSH stimulation of IGFBP-3 transcription is mediated by cAMP via the PKA pathway and requires the P1-3 kinase and likely the MAPK pathways.

Animals↗

PubMed2004

FoxO1a can alter cell cycle progression by regulating the nuclear localization of p27kip in granulosa cells.

Forkhead transcription factors of the FOXO family are important downstream targets of the phosphatidylinositol 3-kinase pathway, which has been shown to play a critical role in cell proliferation and cell survival. Activation of FOXOs can block cellular proliferation and drive cells into a quiescent state. In certain cell types, this cell cycle arrest is dependent on the transcriptional induction of the cell-cycle inhibitor p27kip. In granulosa cells, which go through an exponential growth phase during development of the ovarian follicle, we find that FoxO1a is a key regulator of the G1/S transition in these cells. Overexpression of a dominant-negative version of FoxO1a (Foxo1a-Delta256; a C-terminal truncation mutant that possesses a functional DNA-binding domain, but lacks a transactivation domain) causes a dramatic increase in S-phase cells (>8-fold increase by both DNA content and bromodeoxyuridine incorporation assays). Surprisingly, this is not dependent on transactivation of the p27kip gene. We provide evidence that when FoxO1a activity is impeded, p27kip protein is largely localized to the cytosol, suggesting that FoxO1a blocks cell cycle entry by altering the compartmentalization of p27kip within the cell, increasing its concentration in the nucleus. These studies demonstrate for the first time that FoxO1a can regulate p27kip nuclear localization.

Animals↗

PubMed2004

Insulin-like growth factor-binding protein-3 in porcine ovarian granulosa cells: gene cloning, promoter mapping, and follicle-stimulating hormone regulation.

The role and regulation of IGF-binding protein-3 (IGFBP-3) in the ovary is not fully understood. We cloned and determined the sequence of 12,257 bp of the pig IGFBP-3 gene that includes 4,296 bp of the flanking promoter sequence. The porcine IGFBP-3 promoter sequence shares two highly conserved regions with the human and bovine IGFBP-3 promoters and a mouse DNA clone. The first is a 38 bp region between -1095 and -1058, whereas the second is a 73-bp region between -63 and +10 of the pig sequence. Projected translation of the open reading frame of our sequence gave a peptide sequence identical to that determined by peptide sequencing, but with 27 additional amino acids upstream of this sequence and is highly similar to the human, bovine, rat, and mouse IGFBP-3 peptides. Using RT-PCR we demonstrated that FSH regulates IGFBP-3 mRNA expression in a biphasic manner, with an early induction (maximal at 3 h) and an inhibition at 24 h after FSH treatment. The inhibition at 24 h was not due to changes in IGFBP-3 mRNA stability. A similar pattern of FSH modulation of the IGFBP-3 gene transcription was demonstrated by the reporter activity of granulosa cells transiently transfected with IGFBP-3 promoter constructs. The site for FSH stimulation of the IGFBP-3 gene was localized to the sequence between -61 and -48 relative to the transcription start site. Regulation of IGFBP-3 transcription by FSH suggests a role for IGFBP-3 in follicular development that may be independent of IGF-I.

Animals↗

PubMed2004

Primary, secondary, and tertiary hyperparathyroidism.

Primary, secondary, and tertiary hyperparathyroidism have evolved since their original description. What was once a debilitating disease has now become one with few symptoms on initial presentation. Complications from these disorders have decreased significantly because of earlier detection. Improved management of patients with chronic renal disease has also limited complications among those with secondary and tertiary hyperparathyroidism. Appropriate management is essential even in the early phase of the disorder to minimize the morbidities that may result if left untreated.

Humans↗

PubMed2003

Follicle-stimulating hormone promotes nuclear exclusion of the forkhead transcription factor FoxO1a via phosphatidylinositol 3-kinase in porcine granulosa cells.

The forkhead family of transcription factors is conserved in evolution and known to play critical roles in the regulation of cellular differentiation and proliferation in many systems. The current studies demonstrate for the first time that forkhead homolog in rhabdomyosarcoma (FKHR) (FoxO1a) is expressed in porcine granulosa cells, and FSH stimulates FKHR phosphorylation and regulates its subcellular localization in this system. RT-PCR and Western blot studies demonstrated that FKHR is expressed and showed no change in FKHR message or protein levels in response to FSH (0-6 h). However, [32p]-orthophosphate labeling of cultured granulosa cells revealed robust phosphorylation after FSH treatment for 30 min. In addition, FSH caused nuclear exclusion of FKHR in these cells, apparently through the phosphatidylinositol 3-kinase signal transduction pathway. The cytosolic accumulation of FKHR protein that was observed in FSH-treated cells both by Western blot and immunohistochemistry was blocked when the cells were preincubated with the phosphatidylinositol 3-kinase inhibitor LY294002. Our data also demonstrate that Akt/protein kinase B, an established kinase for FKHR, is phosphorylated in response to FSH treatment. Interestingly, although FKHR was phosphorylated by 30 min after FSH treatment, the time course for Akt phosphorylation was relatively delayed and sustained. Although these studies do not preclude Akt involvement in FSH-stimulated FKHR phosphorylation, they do suggest that other kinases may contribute to rapid signaling to FKHR. Because FKHR has been shown to activate genes involved in apoptosis and growth inhibition, FSH may promote growth and survival by initiating the phosphorylation of FKHR, causing its nuclear exclusion, and reducing its effect as a cell cycle arrest or death-promoting transcription factor.

Animals↗