Organism-Environment Interactions Course Lecture


Coping with Environmental Challenges: A Brief Overview of Cellular and Molecular Actions of Hormones

The environment may influence hormonal changes in organisms. Organisms sense, integrate, and respond to internal and external signals in order to modulate homeostasis, development, and functional processes. These signals are detected at the cellular level and include, but are not limited to, temperature, light, pH, hydration, mechcanial stress, nutrient availability, and quorum sensing.

generic signaling pathway.png
Signals bind to the receptor (typically) on the outer surface of the cell, causing a signal cascade that is propagated through the system. Once the signal is integrated, a response is generated

From lecture

Cell-to-cell communication may be direct, autocrine, paracrine, or endocrine. Cell-to-cell contact is accomplished via gap junctions or mediated by receptor-ligand complexes.

At the biochemical level, gene expression is altered at the level of transcription or translation. Protein-protein interactions are disrupted.
At the physiological level, the activity of the cell changes; the cell may initiate or cease division or differentiation, movement may be induced or reduced, or the cell may undergo apoptosis.

Hormone receptors bind with great specificity.

signals must be detected by cellular receptors.png
From lecture

Lipophilic hormones bind with receptors in the cell nucleus. Able to diffuse or slip past the plasma membrane, they are slow-acting but produce long-lasting effects because they induce changes in gene transcription. Recent studies show that they may also bind with receptors at the membrane surface as well. Hydrophilic hormones bind with receptors on the cell plasma membrane. Once bound, they initiate a signal cascade, whereby the signal is amplified via second messengers. Typically, phosphorylation of a protein occurs.

Stress responses generate epinephrine secretion, causing vasodilation orvasoconstriction in various regions of the body. We know this as the "adrenaline rush". Cortisol, a stress hormone, modulates the body's sensitivity to epinephrine. It also causes the production of glucose, a survival mechanism against starvation. Without cortisol, blood pressure cannot rise. In humans, cortisol levels peak around 6 AM.

G-protein.png
Bockaert and Pin, 1999

G-protein-coupled receptors (GPCRs) are used by an enormous variety of organisms.
-GTP à GDP swapped by alpha subunit, which then disassociates from y- and b-subunits
-GDP-alpha-subunit can interact with other molecules
-G proteins have intrinsic GTPase
à 2nd messengers (variety: cAMP, Ca2+ major ones)
-to get signal across membrane
-amplification
-1 signal : 1 receptor : 1 G-protein : many second messengers

Other methods involve:
-directly affecting ion channels
-tyrosine-kinase-linked receptors
-receptors with intrinsic enzymatic activity, such as guanylate cyclase

Many modules and feedback loops are involved in signal cascades. Thus, there is a great amount of cross-talk between systems.

anatomy of a nuclear hormone receptor.png
From lecture

Not all receptors are found on the membrane. Some can interact with DNA directly.
The hormone binding section binds around greasy bodies. Various parts of the protein have multiple roles, binding with hormones or interacting with hormones to communicate an on/off response.

Members of a family have homologous amino acid sequences and nuclear hormone receptors, which are extremely well-conserved. In frogs, chickens, and humans, the ligand and ligand-binding system is the same.

rxrheterodimer.png
From lecture

Relevant genes are suppressed before any ligands are present, so when the ligand appears and binds to the receptor, the coactivator appears as part of a coactivator complex.
The corepressor works the same way.
This strategy is useful for a wide dynamic of on/off switches (for example, during the metamorphosis of a tadpole into a frog, the limbs are generated before the tail is lost)


Animals as Tools for Environmental Toxicology
alligator.png

Lake Apopka alligators witnessed a 90% population decline in the 1980's.
The remaining alligators were mostly female, but the males mysteriously became feminized.
It turns out there was a pesticide dump containing high levels of DDT
DDT effects = partial to full sex reversal

DDT was discovered to bind to and activate alligator estrogen receptors

First study of estrogen effects on an organism: Rainbow trout vitellogenesis assay
Response elements: signal binds to receptor
Inject male trout with estrogen and use vitellogenin for an estrogen receptor
Result: Induction in all vitellogenin

Thyroid Hormone Endocrine System
*modulatory system, no on/off switch
TH endocrine system.png
Dayan CM and Panicker V (2009) Nat Rev Endocrinol doi:10.1038/nrendo.2009.19

Quantitative high throughput screening for Thyroid Hormone-modulating chemicals in GH3.TRE-LUC cells

Procedure
  • 1536 well format: 1500 cells/well in 4-5 ul media
  • EC50 for T3 0.55 nM
  • Agonist mode (no added T3) and antagonist mode (+ 1nM T3)
  • Assays done over 11-15 concentrations and in triplicate
  • Cell viability assay carried out in parallel

transgenic mouse.png
Use transgenic reporter animals for in vivo screening

Toxicological Sciences 121(2), 207-233 (2011)


In vivo assay for TH: TH-induced Metamorphosis in the African Clawed Frog Xenopus laevis

*Transgenic Xenopus laevis reporter tadpoles may facilitate compound screening

GFP cell model for TH action (in vitro assay)

Procedure

Cells are permeabilized to luciferase (Dr. Furlow's lab uses TRE-luciferase+). The level of luciferase induction can be measured by measuring total luciferase activity.
  • 4-OH PBDE 69 vs. 4-OH PBDE 121
  • Agonist mode - no T3 or very little T3
  • Antagonist mode (compete with agonist) - add 1nM T3
  • Screen cells

Assays are done over 11-15 concentrations and in triplicate, while the cell viability assays are carried out in parallel. The luciferase assay could be verified in parallel with gene expression assays. When the animal is injected with a substrate (estrogen), the luciferase response allows you to visualize the activity.




Related Readings:

  • Miller TC, Sun G, Hasebe T, Fu L, Heimeier RA, Das B, Ishizuya-Oka A, Shi YB (2013) Tissue-Specific Upregulation of MDS/EVI Gene Transcripts in the Intestine by Thyroid Hormone during Xenopus Metamorphosis. PLoS One 8(1):e55585.
  • Tietge JE, Degitz SJ, Haselman JT, Butterworth BC, Korte JJ, Kosian PA, Lindberg-Livingston AJ, Burgess EM, Blackshear PE, Hornung MW (2013) Inhibition of the thyroid hormone pathway in Xenopus laevis by 2-mercaptobenzothiazole. Aquatic Toxicology 126:128-36.
  • Fini JB, Le Mével S, Palmier K, Darras VM, Punzon I, Richardson SJ, Clerget-Froidevaux MS, Demeneix BA (2012) Thyroid hormone signaling in the Xenopus laevis embryo is functional and susceptible to endocrine disruption. Endocrinology 153(10):5068-81.
  • Denver, RJ, Scanlan TS, and JD Furlow (2009) Thyroid hormone receptor subtype specificity for hormone-dependent neurogenesis in Xenopus laevis. Developmental Biology (in press).
  • Johnsen SA, Güngör C, Prenzel T, Riethdorf, S, Riethdorf L, Taniguchi-Ishigaki N, Rau T, Tursun B, Furlow JD, Sauter G, Scheffner M, Pantel K, Gannon F, and I Bach. (2009) Identification of the LIM cofactors CLIM and RLIM as Novel Regulators of Estrogen-Dependent Transcription in Breast Cancer. Cancer Research. 69:128-136
  • Schriks M, Roessig JM, Murk AJ, Furlow JD (2007) Thyroid hormone receptor isoform selectivity of thyroid hormone disrupting compounds quantified with an in vitro reporter gene assay. Environmental Toxicology and Pharmacology 23:320-307.
  • Furlow, J.D. and E.S. Neff (2006) A developmental switch induced by thyroid hormone: Xenopus laevis metamorphosis. Trends in Endocrinolology and Metabolism 17:40-47.

Resources:

Cell signaling: http://www.biochemj.org/csb/default.htm



The Furlow Lab:
The Furlow Lab investigates the control of gene expression by thyroid hormone receptors during metamorphosis in Xenopus laevis (African clawed frog). Other relevant interests pertain to the effect of environmental chemicals in the modulation of thyroid hormone receptor activity; development of synthetic thyromimetic compounds; molecular basis of hormone action during development; analysis of gene expression cascades during morphogenesis; mechanisms of skeletal muscle atrophy and death.

About the Professor:

Dr. J. David Furlow is an Associate Professor at the University of California, Davis, in the Neurobiology, Physiology, and Behavior section of the College of Biological Sciences. He received his bachelor's degree in Biochemistry from the Pennsylvania State University and his Ph.D. in Biochemistry at the University of Wisconsin. Under Dr. Jack Gorski, he did his thesis work on estrogen receptor structure and function. Afterwards, he did his post-doctoral training in Dr. Donald Brown's laboratory at the Carnegie Institution of Washington Department of Embryology.

Research Interests:

Molecular basis of hormone action, particularly during development
Analysis of gene expression cascades during morphogenesis
Mechanisms of skeletal muscle atrophy and death

Teaching Interests:

Endocrinology
Developmental Biology
Molecular biology

Courses taught by Dr. Furlow:

NPB 101 Systemic Physiology - Winter
NPB 128 Comparative Physiology: Endocrinology - Winter
NPB 152 Hormones and Behavior - Spring