Robert Claycomb
Harvard University
Network
Latest external collaboration on country level. Dive into details by clicking on the dots.
Publication
Featured researches published by Robert Claycomb.
Annals of the New York Academy of Sciences | 2008
Dona L. Wong; T.C. Tai; David C. Wong-Faull; Robert Claycomb; Richard Kvetňanský
Stress effects on adrenergic responses in rats were examined in adrenal medulla, the primary source of circulating epinephrine (Epi). Irrespective of duration, immobilization (IMMO) increased adrenal corticosterone to the same extent. In contrast, Epi changed little, suggesting that Epi synthesis replenishes adrenal pools and sustains circulating levels for the heightened alertness and physiological changes required of the “flight or fight” response. IMMO also induced the Epi‐synthesizing enzyme, phenylethanolamine N‐methyltransferase (PNMT). The rise in its mRNA and protein was preceded by increases in Egr‐1 and Sp1 mRNA, protein, and protein‐DNA binding complex formation. With repeated and prolonged stress, PNMT protein did not reflect the magnitude of change in mRNA. The latter suggests that post‐transcriptional, in addition to transcriptional mechanisms, regulate PNMT responses to stress. To further reveal molecular mechanisms underlying stress‐induced changes in adrenergic function, the effects of hypoxia on PNMT promoter‐driven gene expression are being examined in adrenal medulla‐derived PC12 cells. Hypoxia activates the PNMT promoter to increase PNMT promoter‐driven luciferase reporter gene expression and endogenous PNMT in PC12 cells. Induction of both appear mediated via activation of multiple signaling pathways and downstream activation of hypoxia inducible factor and PNMT transcriptional activators, Egr‐1 and Sp1. Hypoxia generates both partially and fully processed forms of PNMT mRNA. The former reportedly is translated into a truncated, nonfunctional protein, and the latter into enzymatically active PNMT. Together, findings suggest that stress increases PNMT gene transcriptional activity but post‐transcriptional regulatory mechanisms limit the biological end‐point of functional PNMT enzyme and, thereby, Epi.
Cellular and Molecular Neurobiology | 2012
Dona L. Wong; T. C. Tai; David C. Wong-Faull; Robert Claycomb; Edward G. Meloni; Karyn M. Myers; William A. Carlezon; Richard Kvetnansky
Epinephrine (Epi), which initiates short-term responses to cope with stress, is, in part, stress-regulated via genetic control of its biosynthetic enzyme, phenylethanolamine N-methyltransferase (PNMT). In rats, immobilization (IMMO) stress activates the PNMT gene in the adrenal medulla via Egr-1 and Sp1 induction. Yet, elevated Epi induced by acute and chronic stress is associated with stress induced, chronic illnesses of cardiovascular, immune, cancerous, and behavioral etiologies. Major sources of Epi include the adrenal medulla and brainstem. Although catecholamines do not cross the blood–brain barrier, circulating Epi from the adrenal medulla may communicate with the central nervous system and stress circuitry by activating vagal nerve β-adrenergic receptors to release norepinephrine, which could then stimulate release of the same from the nucleus tractus solitarius and locus coeruleus. In turn, the basal lateral amygdala (BLA) may activate to stimulate afferents to the hypothalamus, neocortex, hippocampus, caudate nucleus, and other brain regions sequentially. Recently, we have shown that repeated IMMO or force swim stress may evoke stress resiliency, as suggested by changes in expression and extinction of fear memory in the fear-potentiated startle paradigm. However, concomitant adrenergic changes seem stressor dependent. Present studies aim to identify stressful conditions that elicit stress resiliency versus stress sensitivity, with the goal of developing a model to investigate the potential role of Epi in stress-associated illness. If chronic Epi over expression does elicit illness, possibilities for alternative therapeutics exist through regulating stress-induced Epi expression, adrenergic receptor function and/or corticosteroid effects on Epi, adrenergic receptors and the stress axis.
Journal of Neurochemistry | 2007
T. C. Tai; Robert Claycomb; Brenda J. Siddall; Rose Ann Bell; Richard Kvetnansky; Dona L. Wong
Immobilization (IMMO) stress was used to examine how stress alters the stress hormone epinephrine (EPI) in the adrenal medulla in vivo. In rats subjected to IMMO for 30 or 120 min, adrenal corticosterone increased to the same extent. In contrast, EPI changed very little, suggesting that EPI synthesis replenishes adrenal pools and sustains circulating levels for the heightened alertness and physiological responses of the ‘flight or fight’ response. In part, stress activates EPI via the phenylethanolamine N‐methyltransferase (PNMT) gene as single or repeated IMMO elevated PNMT mRNA. The rise in PNMT mRNA was preceded by induction of the PNMT gene activator, Egr‐1, with increases in Egr‐1 mRNA, protein, and protein–DNA binding complex apparent. IMMO also evoked changes in Sp1 mRNA, protein, and Sp1–DNA complex formation, although for chronic IMMO changes were not entirely coincident. In contrast, glucocorticoid receptor and AP‐2 mRNA, protein, and protein–DNA complex were unaltered. Finally, IMMO stress elevated PNMT protein. However, with seven daily IMMOs for 120 min and delayed killing, protein stimulation did not attain the highly elevated levels expected based on mRNA changes. The latter may perhaps suggest initiation of adrenergic desensitization to prolonged and repeated IMMO stress and/or dissociation of transcriptional and post‐transcriptional regulatory mechanisms.
Annals of the New York Academy of Sciences | 2004
Dona L. Wong; T. C. Tai; David C. Wong-Faull; Robert Claycomb; Richard Kvetnansky
Abstract: Cortisol and epinephrine released in response to stress are replenished via activation of the hypothalamic‐pituitary‐adrenal (HPA or stress) axis. Immobilization (IMMO) stress in rats stimulates epinephrine production in part via the gene encoding the epinephrine‐synthesizing enzyme phenylethanolamine N‐methyltransferase (PNMT). PNMT mRNA rose up to 7.0‐fold with acute or chronic stress. Two transcription factors mediating stress induction of the PNMT gene are the glucocorticoid receptor (GR) and Egr‐1, which interact with −533, −759, and −773 bp, and −165 bp binding sites in the rat PNMT promoter, respectively. To identify molecular mechanisms involved, effects of hypoxic stress on PNMT promoter activity were examined in PC12 cells transfected with the PNMT promoter‐luciferase reporter gene construct pGL3RP893. Oxygen reduction to 5% increased PNMT promoter‐driven luciferase expression, with maximum activity at 6 h. Pretreatment of the cells with protein kinase A (PKA) and protein kinase C (PKC) inhibitors, H‐89 and GF109203X, respectively, attenuated the rise in luciferase. Similarly, PKA‐deficient PC12 cells transfected with pGL3RP893 and exposed to hypoxia also showed attenuated PNMT promoter‐driven luciferase expression. Mutation of the Egr‐1 binding site completely prevented PNMT promoter activation, indicating that Egr‐1 is essential to the stress response. Consistent with this result, hypoxia increased Egr‐1 protein. Hypoxia also increased endogenous PNMT mRNA. However, a shift to intron‐retaining mRNA from which truncated, nonfunctional protein is produced, occurred, suggesting that posttranscriptional regulation may be an important genetic mechanism controlling adrenergic expression and hence, epinephrine, during stress.
Journal of Neurochemistry | 2009
T. C. Tai; David C. Wong-Faull; Robert Claycomb; Dona L. Wong
Sustaining epinephrine‐elicited behavioral and physiological responses during stress requires replenishment of epinephrine stores. Egr‐1 and Sp1 contribute by stimulating the gene encoding the epinephrine‐synthesizing enzyme, phenylethanolamine N‐methyltransferase (PNMT), as shown for immobilization stress in rats in adrenal medulla and for hypoxic stress in adrenal medulla‐derived PC12 cells. Hypoxia (5% O2) also activates hypoxia inducible factor (HIF) 1α, increasing mRNA, nuclear protein and nuclear protein/hypoxia response element binding complex formation. Hypoxia and HIF1α over‐expression also elevate PNMT promoter‐driven luciferase activity in PC12 cells. Hypoxia may be limiting as HIF1α over‐expression increases luciferase expression to no greater extent than oxygen reduction alone. HIF1α inducers CoCl2 or deferoxamine elevate luciferase as well. PC12 cells harboring a HIF1α expression construct show markedly higher levels of Egr‐1 and Sp1 mRNA and nuclear protein and PNMT mRNA and cytoplasmic protein. Inactivation of Egr‐1 and Sp1 binding sites in the proximal −893 bp of PNMT promoter precludes HIF1α stimulation while a potential hypoxia response element (−282 bp) in the promoter shows weak HIF1α affinity at best. These findings are the first to suggest that hypoxia activates the proximal rat PNMT promoter primarily via HIF1α induction of Egr‐1 and Sp1 rather than by co‐activation by Egr‐1, Sp1 and HIF1α. In addition, the rise in HIF1α protein leading to Egr‐1 and Sp1 stimulation of PNMT appears to include HIF1α gene activation rather than simply prevention of HIF1α proteolytic degradation.
Brain Research | 2010
T. C. Tai; David C. Wong-Faull; Robert Claycomb; Dona L. Wong
Hypoxia is shown to regulate the stress hormone epinephrine through its biosynthesis by phenylethanolamine N-methyltransferase (PNMT) via PNMT gene activation and transcription factors Egr-1 and Sp1 in adrenal medulla-derived PC12 cells. Moderate hypoxia (5% oxygen) markedly stimulates PNMT promoter-driven luciferase activity in the cells. Hypoxia increases Egr-1 and Sp1 mRNA and nuclear protein content and Egr-1 and Sp1 protein-DNA binding complex formation. Subsequent to transcription factor induction, endogenous PNMT mRNA and protein also increase. Egr-1 and Sp1 binding site inactivation or Egr-1 and Sp1 siRNA inhibit PNMT promoter stimulation by hypoxia. Hypoxia elevates protein kinase A (PKA), phospholipase C (PLC), phosphoinositide 3-kinase, protein kinase C, ERK1/2 mitogen-activated protein kinase and p38 mitogen-activated protein kinase expression while selective inhibitors of these signaling enzymes abrogate hypoxic induction of the PNMT promoter and the rise in Egr-1, Sp1 and PNMT mRNA and protein. PC12 cells lacking PKA or PLCgamma-1 show significant reduction in PNMT promoter activation by hypoxia. Signaling inhibitors do not affect these responses or reduce hypoxic induction of the PNMT promoter to a lesser extent. Findings suggest that Egr-1 and Sp1 through synergistic interaction are critical transcriptional activators for hypoxic stress-regulated adrenergic function controlled via cAMP/PKA and PLC signaling. Identification of Sp1 as a mediator of hypoxia-induced transcriptional activation of PNMT has not been previously been shown. The effects of hypoxia on PNMT and thereby epinephrine may have important ramifications for the stress hormone epinephrine, its ability to regulate behavioral and physiological processes associated with stress and stress-elicited illness.
Molecular Pharmacology | 2006
T. C. Tai; David C. Wong-Faull; Robert Claycomb; Dona L. Wong
The mechanism by which nerve growth factor (NGF) regulates adrenergic expression was examined in PC-12 cells transfected with a rat phenylethanolamine N-methyl-transferase (PNMT) promoter-luciferase reporter gene construct pGL3RP893. NGF treatment increased PNMT promoter-driven luciferase activity in a dose- and time-dependent manner. Induction was attenuated by inhibition of the extracellular signal-regulated kinase mitogen-activated protein kinase (MAPK) pathway (∼60%) but not by inhibition of the protein kinase A (PKA), protein kinase C, phosphoinositol kinase, or p38 MAPK pathways. Deletion PNMT promoter-luciferase reporter gene constructs showed that the NGF-responsive sequences lay within the proximal -392 base pairs (bp) of PNMT promoter, wherein binding elements for Egr-1 (-165 bp) and Sp1 (-48 bp) reside. Western analysis further showed that NGF increased nuclear levels of Egr-1, but not Sp1 or the catalytic subunit of PKA. Gel mobility shift assays showed increased potential for Egr-1, but not Sp1, protein-DNA binding complex formation. Mutation of either the Egr-1 or Sp1 binding sites in the PNMT promoter attenuated NGF activation. NGF, combined with pituitary adenylyl cyclase-activating protein (PACAP), another PNMT transcriptional activator, cooperatively stimulated PNMT promoter driven-luciferase activity beyond levels observed with either neurotrophin alone. Finally, post-transcriptional control seems to be another important mechanism by which neurotrophins regulate the adrenergic phenotype. NGF, PACAP, and a combination of the two stimulated both intron-retaining and intronless PNMT mRNA and PNMT protein, but to different extents.
Journal of Neurochemistry | 2010
Tze Chun Tai; David C. Wong-Faull; Robert Claycomb; Jennifer L. Aborn; Dona L. Wong
J. Neurochem. (2010) 115, 1195–1205.
Molecular Pharmacology | 2002
T. C. Tai; Robert Claycomb; Song Her; A. K. Bloom; Dona L. Wong
Molecular Pharmacology | 2003
Song Her; Robert Claycomb; T. C. Tai; Dona L. Wong