Five degrees-of-freedom mechanical arm with remote center of motion (RCM) device for volumetric optical coherence tomography (OCT) retinal imaging.

Handheld optical coherence tomography (HH-OCT) is gaining popularity for diagnosing retinal diseases in neonates (e.g. retinopathy of prematurity). Diagnosis accuracy is degraded by hand tremor and patient motion when using commercially available handheld retinal OCT probes. This work presents a low-cost arm designed to address ergonomic challenges of holding a commercial OCT probe and alleviating hand tremor. Experiments with a phantom eye show enhanced geometric uniformity and volumetric accuracy when obtaining OCT scans with our device compared to handheld imaging approaches. An in-vivo porcine volumetric image was also obtained with the mechanical arm demonstrating clinical deployability.

Morphological and functional changes in the rat retina associated with 2 months of intermittent moderate intraocular pressure elevation.

Morphological and functional changes in the rat retina and optic nerve head (ONH), associated with 8 weeks of intermittent moderately elevated intraocular pressure (IOP) were measured with a combined ultrahigh resolution optical coherence tomography (UHR-OCT) and electroretinography (ERG) system. The IOP of male Sprague-Dawley rats was raised in one eye to ~35 mmHg for 1 hour/day on 6 days each week using vascular loops. Single-flash ERG traces and volumetric UHR-OCT images of the ONH were acquired from both eyes before, during and after IOP elevations at weeks 1, 5 and 9 of the study. The UHR-OCT images showed depression of the posterior eye around the ONH during the IOP elevations, the magnitude of which increased significantly from week 1 to week 9 (p = 0.01). The ERG a-wave and b-wave amplitudes increased temporarily during IOP elevations and returned to normal ~30 minutes after loop removal. Recurrent intermittent IOP spikes caused > 30% decrease in the ERG a-wave and b-wave amplitudes measured during the IOP elevations over the course of 2 months. This study suggests that recurrent, relatively short-duration IOP spikes for extended period of time are associated with peri-ONH tissue hypercompliance and reduced retinal functional response to visual stimulation during acute IOP elevation.

Short-Term Moderately Elevated Intraocular Pressure Is Associated With Elevated Scotopic Electroretinogram Responses.

PURPOSE: Moderately elevated intraocular pressure (IOP) is a risk factor for open-angle glaucoma. Some patients suffer glaucoma despite clinically measured normal IOPs. Fluctuations in IOP may have a significant role since IOPs are higher during sleep and inversion activities. Controlled transient elevations of IOPs in rats over time lead to optic nerve structural changes that are similar to the early changes observed in constant chronic models of glaucoma. Because early intervention decreases glaucoma progression, this study was done to determine if early physiological changes to the retina could be detected with noninvasive electrophysiological and optical imaging tests during moderately elevated IOP. METHODS: Intraocular pressures were raised to moderately high levels (35 mm Hg) in one eye of Sprague-Dawley rats while the other (control) eye was untreated. One group of rats underwent scotopic threshold response (STR) and electroretinogram (ERG) testing, while another 3 groups underwent optical coherence tomography (OCT) imaging, Western blot, or histologic evaluation. RESULTS: The amplitudes of the STR and ERG responses in eyes with moderately elevated IOPs were enhanced compared to the values before IOP elevation, and compared to untreated contralateral eyes. Structural changes to the optic nerve also occurred during IOP elevation. CONCLUSIONS: Although ischemic IOP elevations are well-known to globally reduce components of the scotopic ERG, acute elevation in rats to levels often observed in untreated glaucoma patients caused an increase in these parameters. Further exploration of these phenomena may be helpful in better understanding the mechanisms mediating early retinal changes during fluctuating or chronically elevated IOP.

Optic nerve sheath fenestration using a Raman-shifted alexandrite laser.

BACKGROUND AND OBJECTIVE: Optic nerve sheath fenestration is an established procedure for relief of potentially damaging overpressure on the optic nerve resulting from idiopathic intracranial hypertension. Prior work showed that a mid-IR free-electron laser could be delivered endoscopically and used to produce an effective fenestration. This study evaluates the efficacy of fenestration using a table-top mid-IR source based on a Raman-shifted alexandrite (RSA) laser. STUDY DESIGN/MATERIALS AND METHODS: Porcine optic nerves were ablated using light from an RSA laser at wavelengths of 6.09, 6.27, and 6.43 µm and pulse energies up to 3 mJ using both free-space and endoscopic beam delivery through 250-µm I.D. hollow-glass waveguides. Waveguide transmission was characterized, ablation thresholds and etch rates were measured, and the efficacy of endoscopic fenestration was evaluated for ex vivo exposures using both optical coherence tomography and histological analysis. RESULTS: Using endoscopic delivery, the RSA laser can effectively fenestrate porcine optic nerves. Performance was optimized at a wavelength of 6.09 µm and delivered pulse energies of 0.5-0.8 mJ (requiring 1.5-2.5 mJ to be incident on the waveguide). Under these conditions, the ablation threshold fluence was 0.8 ± 0.2 J/cm(2) , the ablation rate was 1-4 µm/pulse, and the margins of ablation craters showed little evidence of thermal or mechanical damage. Nonetheless, nominally identical exposures yielded highly variable ablation rates. This led to fenestrations that ranged from too deep to too shallow-either damaging the underlying optic nerve or requiring additional exposure to cut fully through the sheath. Of 48 excised nerves subjected to fenestration at 6.09 µm, 16 ex vivo fenestrations were judged as good, 23 as too deep, and 9 as too shallow. CONCLUSIONS: Mid-IR pulses from the RSA laser, propagated through a flexible hollow waveguide, are capable of cutting through porcine optic nerve sheaths in surgically relevant times with reasonable accuracy and low collateral damage. This can be accomplished at wavelengths of 6.09 or 6.27 µm, with 6.09 µm slightly preferred. The depth of ex vivo fenestrations was difficult to control, but excised nerves lack a sufficient layer of cerebrospinal fluid that would provide an additional margin of safety in actual patients.

Efficacy and predictability of soft tissue ablation using a prototype Raman-shifted alexandrite laser.

Previous research showed that mid-infrared free-electron lasers could reproducibly ablate soft tissue with little collateral damage. The potential for surgical applications motivated searches for alternative tabletop lasers providing thermally confined pulses in the 6- to-7-µm wavelength range with sufficient pulse energy, stability, and reliability. Here, we evaluate a prototype Raman-shifted alexandrite laser. We measure ablation thresholds, etch rates, and collateral damage in gelatin and cornea as a function of laser wavelength (6.09, 6.27, or 6.43 µm), pulse energy (up to 3 mJ/pulse), and spot diameter (100 to 600 µm). We find modest wavelength dependence for ablation thresholds and collateral damage, with the lowest thresholds and least damage for 6.09 µm. We find a strong spot-size dependence for all metrics. When the beam is tightly focused (~100-µm diameter), ablation requires more energy, is highly variable and less efficient, and can yield large zones of mechanical damage (for pulse energies>1 mJ). When the beam is softly focused (~300-µm diameter), ablation proceeded at surgically relevant etch rates, with reasonable reproducibility (5% to 12% within a single sample), and little collateral damage. With improvements in pulse-energy stability, this prototype laser may have significant potential for soft-tissue surgical applications.

Miniature forward-imaging B-scan optical coherence tomography probe to guide real-time laser ablation.

BACKGROUND AND OBJECTIVE: Investigations have shown that pulsed lasers tuned to 6.1 µm in wavelength are capable of ablating ocular and neural tissue with minimal collateral damage. This study investigated whether a miniature B-scan forward-imaging optical coherence tomography (OCT) probe can be combined with the laser to provide real-time visual feedback during laser incisions. STUDY DESIGN/METHODS AND MATERIALS: A miniature 25-gauge B-scan forward-imaging OCT probe was developed and combined with a 250 µm hollow-glass waveguide to permit delivery of 6.1 µm laser energy. A gelatin mixture and both porcine corneal and retinal tissues were simultaneously imaged and lased (6.1 µm, 10 Hz, 0.4-0.7 mJ) through air. The ablation studies were observed and recorded in real time. The crater dimensions were measured using OCT imaging software (Bioptigen, Durham, NC). Histological analysis was performed on the ocular tissues. RESULTS: The combined miniature forward-imaging OCT and mid-infrared laser-delivery probe successfully imaged real-time tissue ablation in gelatin, corneal tissue, and retinal tissue. Application of a constant number of 60 pulses at 0.5 mJ/pulse to the gelatin resulted in a mean crater depth of 123 ± 15 µm. For the corneal tissue, there was a significant correlation between the number of pulses used and depth of the lased hole (Pearson correlation coefficient = 0.82; P = 0.0002). Histological analysis of the cornea and retina tissues showed discrete holes with minimal thermal damage. CONCLUSIONS: A combined miniature OCT and laser-delivery probe can monitor real-time tissue laser ablation. With additional testing and improvements, this novel instrument has the future possibility of effectively guiding surgeries by simultaneously imaging and ablating tissue.

Raman-shifted alexandrite laser for soft tissue ablation in the 6- to 7-µm wavelength range.

Prior work with free-electron lasers (FELs) showed that wavelengths in the 6- to 7-µm range could ablate soft tissues efficiently with little collateral damage; however, FELs proved too costly and too complex for widespread surgical use. Several alternative 6- to 7-µm laser systems have demonstrated the ability to cut soft tissues cleanly, but at rates that were much too low for surgical applications. Here, we present initial results with a Raman-shifted, pulsed alexandrite laser that is tunable from 6 to 7 µm and cuts soft tissues cleanly-approximately 15 µm of thermal damage surrounding ablation craters in cornea-and does so with volumetric ablation rates of 2-5 × 10(-3) mm(3)/s. These rates are comparable to those attained in prior successful surgical trials using the FEL for optic nerve sheath fenestration.

Identification of genes, including the gene encoding p27Kip1, regulated by serine 276 phosphorylation of the p65 subunit of NF-kappaB.

Phosphorylation of the p65 subunit of NF-kappaB is required for its transcriptional activity. Recent reports show that phosphorylation of p65 at serine 276 regulates only a subset of genes, such as those encoding IL-6, IL-8, Gro-beta, and ICAM-1. In order to identify additional genes regulated by serine 276 phosphorylation, HepG2 hepatoma cells were infected with adenoviruses encoding either wild-type p65 or the S276A mutant of p65, followed by DNA microarray analysis. The results show that mutation of serine 276 affected the expression of several genes that encode proteins involved in cell cycle regulation, signal transduction, transcription, and metabolism. Notably, expression of S276A increased the mRNA and protein level of p27, a cell cycle inhibitory protein, which led to an increased association of p27 with cdk2, and inhibition of cdk2 activity. Furthermore, while wild-type NF-kappaB is known to increase cell proliferation in a number of different cancer cell lines, our data shows that S276A inhibited cell proliferation. Evidence is mounting that NF-kappaB plays a pivotal role in oncogenesis. Therapeutic agents that regulate the phosphorylation of serine 276 and p27 gene expression, therefore, may be useful as anti-cancer agents in the future.

15-deoxy-delta12,14-prostaglandin J2 attenuates endothelial-monocyte interaction: implication for inflammatory diseases.

BACKGROUND: The Infiltration of leukocytes across the brain endothelium is a hallmark of various neuroinflammatory disorders. Under inflammatory conditions, there is increased expression of specific cell adhesion molecules (CAMs) on activated vascular endothelial cells which increases the adhesion and infiltration of leukocytes. TNFalpha is one of the major proinflammatory cytokines that causes endothelial dysfunction by various mechanisms including activation of transcription factor NF-kappaB, a key transcription factor that regulates expression of CAMs. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear hormone superfamily of ligand-activated transcriptional factors. 15-deoxy-delta 12, 14-prostaglandin J2 (15d-PGJ2) is a well recognized natural ligand of PPARgamma and possesses anti-inflammatory properties both in vitro and in vivo. This study aims to elucidate the mechanism of 15-PGJ2 on the adhesion of mononuclear cells to activated endothelial cells. METHODS: To delineate the signaling pathway of 15d-PGJ2 mediated effects, we employed an in vitro adhesion assay model of endothelial-monocyte interaction. Expression of CAMs was examined using flow cytometry and real time PCR techniques. To define the mechanism of 15d-PGJ2, we explored the role of NF-kappaB by EMSA (Electrophoretic Mobility Shift Assay) gels, NF-kappaB reporter and p65-transcriptional activities by transient transfection in the brain-derived endothelial cell line (bEND.3). RESULTS: Using an in vitro adhesion assay model, we demonstrate that 15d-PGJ2 inhibits TNFalpha induced monocyte adhesion to endothelial cells, which is mediated by downregulation of endothelial cell adhesion molecules in a PPARgamma independent manner. 15d-PGJ2 modulated the adhesion process by inhibiting the TNFalpha induced IKK-NF-kappaB pathway as evident from EMSA, NF-kappaB reporter and p65 mediated transcriptional activity results in bEND.3 cells. CONCLUSION: These findings suggest that 15d-PGJ2 inhibits inflammation at multiple steps and thus is a potential therapeutic target for various inflammatory diseases.

GSNO attenuates EAE disease by S-nitrosylation-mediated modulation of endothelial-monocyte interactions.

S-Nitrosoglutathione (GSNO) is an endogenous nitric oxide carrier and recently, has been documented for its anti-inflammatory effects in rat model of cerebral ischemia (Khan et al. (2005) J Cereb Blood Flow Metab 25:177-192). Here, we explored the neuroprotective effects mediated by GSNO in Lewis rat model of EAE and its mechanism of action using in vitro model of monocyte-endothelial cell interaction. Oral administration of GSNO attenuated the clinical disease course in EAE animals by inhibiting the infiltration of vascular immune cells in the CNS that subsequently led to the reduction in the expression of proinflammatory cytokines and consequently limited demyelination. Based on the inhibition in infiltration of immune cells, we hypothesized that GSNO modulated endothelial cell activation that led to reduce cellular infiltration in the CNS. Using an in vitro model, we established that GSNO inhibited monocyte adhesion to the activated endothelial cell, which was mediated by down regulation of endothelial cell adhesion molecules (CAMs). The mechanism by which GSNO modulated CAMs expression appeared to be via S-nitrosylation of p65, which consequently inhibited nuclear factor kappa B (NF-kappaB) activation in endothelial cells. These observations suggest that GSNO exerts its protective effects in EAE by inhibition of cellular infiltration into the CNS by S-nitrosylation of p65, thereby modulating NF-kappaB-CAMs pathway in endothelial cells.

T-bet is essential for the progression of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is mediated by myelin-specific CD4+ T helper 1 (Th1) cells, while recovery from the disease is associated with the presence of Th2 cells. Here we used animals with targeted deletion of the T-bet gene to determine its role in the progression of EAE. T-bet regulates the production of interferon-gamma (IFN-gamma) in CD4+ and natural killer cells, and CD4+ T cells from T-bet-deficient mice were unable to differentiate into a Th1 phenotype. Moreover BALB/c mice deficient in T-bet were resistant to the induction of EAE disease, with minimal inflammatory infiltrates in the central nervous system. These mice were resistant to EAE induction even when PLP(180-199) peptide specific effector T cells from BALB/c wild type were transferred to BALB/c T-bet-deficient mice. This resistance to EAE is may be caused by the production of the anti-inflammatory cytokine interleukin-10 (IL-10) from the spleen cells upon ex vivo stimulation with PLP(180-199) peptide and in vivo presence in the central nervous system. There was no difference in the recall responses in spleen cells from T-bet-deficient and wild type mice; however, less secretion of IFN-gamma was observed from primed splenocytes. The expression of IFN-gamma was less in the central nervous system of T-bet-deficient mice whereas IL-10 was significantly higher in T-bet-deficient as compared to wild type mice. These data indicate that T-bet genes play a critical role in maintaining the encephalitogenic nature of CD4+ T cells in autoimmune responses during EAE disease progression.

5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside attenuates experimental autoimmune encephalomyelitis via modulation of endothelial-monocyte interaction.

Experimental autoimmune encephalomyelitis (EAE) is a model for studying multiple sclerosis (MS), a chronic demyelinating disorder of the CNS. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), an activator of AMP-activated protein kinase (AMPK), has been reported to show antiinflammatory and immunomodulatory effects in various models of inflammation. Recently, we have reported AICAR-mediated attenuation of active and passive EAE in mouse model [Nath et al. (2005) J. Immunol. 175:566-574]. Here we used a rat model of acute EAE to show antiinflammatory effects of AICAR after daily treatment starting at onset of the disease. By maintaining the blood-brain barrier (BBB), AICAR-administered animals showed lower clinical scores compared with untreated EAE animals. AICAR inhibited the infiltration of inflammatory cells across the BBB, resulting in lowered expression of proinflammatory mediators in the CNS and protection from severe demyelination. By using in vitro model of endothelial-leukocyte interaction, we showed that AICAR inhibited adhesion of monocytes to tumor necrosis factor-alpha-activated endothelial cells. One of the mechanisms of this action is through down-regulation of expression of endothelial cell adhesion molecules via modulation of nuclear factor kappaB activation. The data suggest that AICAR attenuates EAE progression by limiting infiltration of leukocytes across the BBB, thereby controlling the consequent inflammatory reaction in the CNS.

5-aminoimidazole-4-carboxamide ribonucleoside: a novel immunomodulator with therapeutic efficacy in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is a Th1-mediated inflammatory demyelinating disease of the CNS. AMP-activated protein kinase was reported recently to have anti-inflammatory activities by negatively regulating NF-kappaB signaling. In this study, we investigated the prophylactic and therapeutic efficacy of an AMP-activated protein kinase activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), in active and passive EAE induced by active immunization with PLP(139-151) or MOG(35-55) and in adoptive transfer of PLP(139-151)-sensitized T cells, respectively. In vivo treatment with AICAR exerted both prophylactic and therapeutic effects on EAE, attenuating the severity of clinical disease. The anti-inflammatory effects of AICAR were associated with the inhibition of the Ag-specific recall responses and inhibition of the Th1-type cytokines IFN-gamma and TNF-alpha, whereas it induced the production of Th2 cytokines IL-4 and IL-10. Treatment of PLP(139-151)-specific T cells in vitro with AICAR decreased their expression of T-bet in response to IL-12, a Th1 transcription factor, whereas in response to IL-4, it induced the expression and phosphorylation of Th2 transcription factors GATA3 and STAT6, respectively. Moreover, treatment of APCs in vitro with AICAR inhibited their capability to present the proteolipid protein peptide to PLP(139-151)-specific T cells. In an irrelevant Th1-mediated, OT-2 TCR transgenic mouse model, AICAR impaired in vivo Ag-specific expansion of CD4(+) T cells. Together, these findings show for the first time that AICAR is a novel immunomodulator with promising beneficial effects for the treatment of multiple sclerosis and other Th1-mediated inflammatory diseases.

Inhibition of phosphoinositide 3 kinase-Akt (protein kinase B)-nuclear factor-kappa B pathway by lovastatin limits endothelial-monocyte cell interaction.

Integrity of the blood-brain barrier is essential for the normal functioning of CNS. Its disruption contributes to the pathobiology of various inflammatory neurodegenerative disorders. We have shown that the HMG-CoA reductase inhibitor (lovastatin) attenuated experimental autoimmune encephalomyelitis (EAE, an inflammatory disease of CNS) in rodents by inhibiting the infiltration of mononuclear cells into the CNS. Here, using an in vitro system, we report that lovastatin inhibits endothelial-monocyte cell interaction by down-regulating the expression of vascular cell adhesion molecule-1 and E-selectin by inhibiting the phosphoinositide 3 kinase (PI3-kinase)/protein kinase B (Akt)/nuclear factor-kappa B (NF-kappaB) pathway in endothelial cells. It inhibits tumor necrosis factor alpha (TNFalpha)-induced PI3-kinase, Akt and NF-kappaB activation in these cells. Co-transfection of constitutively active forms of PI3-kinase and Akt reversed the lovastatin-mediated inhibition of TNFalpha-induced adhesion, as well as activation of NF-kappaB, indicating the involvement of the PI3-kinase/Akt pathway in the interaction of adhesion molecules and the process of adhesion. This study reports that lovastatin down-regulates the pathway affecting the expression and interaction of adhesion molecules on endothelial cells, which in turn restricts the migration and infiltration of mononuclear cells thereby attenuating the pathogenesis of inflammatory diseases.

An extract of Syzygium aromaticum represses genes encoding hepatic gluconeogenic enzymes.

Insulin action is impaired in diabetic patients, which leads to increased hepatic glucose production. Plants and herbs have been used for medicinal purposes, including the treatment of diabetes, for centuries. Since dietary management is a starting point for the treatment of diabetes, it is important to recognize the effect of plant-based compounds on tissues that regulate glucose metabolism, such as the liver. In a recent study, several herbs and spices were found to increase glucose uptake into adipocytes, an insulin-like effect. Our data reveal that Syzygium aromaticum (L.) Merrill and Perry (Myrtaceae) (commonly referred to as clove) extract acts like insulin in hepatocytes and hepatoma cells by reducing phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) gene expression. Much like insulin, clove-mediated repression is reversed by PI3K inhibitors and N-acetylcysteine (NAC). A more global analysis of gene expression by DNA microarray analysis reveals that clove and insulin regulate the expression of many of the same genes in a similar manner. These results demonstrate that consumption of certain plant-based diets may have beneficial effects for the treatment of diabetes and indicate a potential role for compounds derived from clove as insulin-mimetic agents.

Potential targets of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor for multiple sclerosis therapy.

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors or statins are newly identified immunomodulators. In vivo treatment of SJL/J mice with lovastatin reduced the duration and clinical severity of active and passive experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Lovastatin induced the expression of GATA3 and the phosphorylation of STAT6, whereas it inhibited tyrosine phosphorylation of Janus kinase 2, tyrosine kinase 2, and STAT4. Inhibition of the Janus kinase-STAT4 pathway by lovastatin modulated T0 to Th1 differentiation and reduced cytokine (IFN-gamma and TNF-alpha) production, thus inducing Th2 cytokines (IL-4, IL-5, and IL-10). It inhibited T-bet (T box transcription factor) and NF-kappaB in activated T cells and significantly reduced infiltration of CD4- and MHC class II-positive cells to CNS. Further, it stabilized IL-4 production and GATA-3 expression in differentiated Th2 cells, whereas in differentiated Th1 cells it inhibited the expression of T-bet and reduced the production of IFN-gamma. Moreover, lovastatin-exposed macrophage and BV2 (microglia) in allogeneic MLRs induced the production of the anti-inflammatory cytokine IL-10. These observations indicate that the anti-inflammatory effects of lovastatin are mediated via T cells as well as APCs, because it modulates the polarization patterns of naive T cell activation in an APC-independent system. Together, these findings reveal that lovastatin may have possible therapeutic value involving new targets (in both APCs and T cells) for the treatment of multiple sclerosis and other inflammatory diseases.

Elements of the glucocorticoid and retinoic acid response units are involved in cAMP-mediated expression of the PEPCK gene.

Although many genes are regulated by the concerted action of several hormones, hormonal signaling to gene promoters has generally been studied one hormone at a time. The phosphoenolpyruvate carboxykinase (PEPCK) gene is a case in point. Transcription of this gene is induced by glucagon (acting by the second messenger, cAMP), glucocorticoids, and retinoic acid, and it is dominantly repressed by insulin. These hormonal responses require the presence of different hormone response units (HRUs), which consist of constellations of DNA elements and associated transcription factors. These include the glucocorticoid response unit (GRU), cAMP response unit (CRU), retinoic acid response unit (RARU), and the insulin response unit. HRUs are known to have functional overlap. In particular, the cAMP response element of the CRU is also a component of the GRU. The purpose of this study was to determine whether known GRU or RARU elements or transcription factors function as components of the CRU. We show here that the glucocorticoid accessory factor binding site 1 and glucocorticoid accessory factor binding site 3 elements, which are components of both the GRU and RARU, are an important part of the CRU. Furthermore, we find that the transcription factor, chicken ovalbumin upstream promoter-transcription factor, and two coactivators, cAMP response element-binding protein-binding protein and steroid receptor coactivator-1, participate in both the cAMP and glucocorticoid responses. This provides a further illustration of how the PEPCK gene promoter integrates different hormone responses through overlapping HRUs that utilize some of the same transcription factors and coactivators.

A point mutation of the AF2 transactivation domain of the glucocorticoid receptor disrupts its interaction with steroid receptor coactivator 1.

Glucocorticoids cause a 10-fold increase in hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene transcription through two low affinity glucocorticoid receptor (GR) binding sites and a complex array of accessory factor DNA elements and associated proteins. To analyze how co-activators interact with the GR in this context, we took advantage of the C656G GR mutant that binds ligand with very high affinity. This GR activates PEPCK gene transcription at a 500-fold lower dexamethasone concentration than does wild type GR. Transfected C656G GR containing additional mutations or deletions was tested on PEPCK gene expression in H4IIE hepatoma cells. We found that the AF2 domain is the only one of the three defined transactivation domains in GR that is required for PEPCK gene expression and that mutation of this domain disrupts the direct interaction of GR with steroid receptor coactivator 1 (SRC-1). These data help define the functional interaction between GR and SRC-1 and further define the role of the GR in glucocorticoid-mediated expression of the PEPCK gene.