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Intramural Papers of the Month

By Laura Hall and Thaddeus Schug
December 2009

Zinc Finger RNA-Binding Protein Zfp36l2 Critical in Hematopoiesis

A collaborative effort of researchers from several institutions led by NIEHS scientists generated mice completely deficient in the zinc finger protein 36, C3H type-like 2 (Zfp36l2). The knock-out (KO) mice died within two weeks and showed very low levels of all types of blood cells, the hematopoietic cells.

The authors found that the definitive multi-lineage and lineage-committed hematopoietic progenitors that occur in the yolk sac of developing embryos from day 11.5 and in fetal liver from day 14.5 were significantly decreased in KO mice compared to wild type. The results indicated that Zfp36l2 played a critical role in the development of the hematopoietic system.

Zfp36l2 is a member of a family of proteins containing zinc fingers - protein domains that bind a zinc ion which help stabilize protein folds. Many zinc finger protein families exist in the mammalian genome and their members can bind to DNA, RNA, other proteins and lipids and are involved in many different cellular processes.

Another member of the ZFP36 family, tristetraprolin, is able to destabilize certain messenger RNAs (mRNAs) by binding to adenine-uridine-rich elements located in the 3' untranslated region. Destabilizing the mRNA coding for a protein prevents the protein from being made, a form of post-transcriptional regulation. The authors suggest that Zfp36l2 may also regulate mRNA encoding proteins critical to hematopoiesis in a similar way.

Citation: Stumpo DJ, Broxmeyer HE, Ward T, Cooper S, Hangoc G, Chung YJ, Shelley WC, Richfield EK, Ray MK, Yoder MC, Aplan PD, Blackshear PJ. ( NIEHS 2009. Targeted disruption of Zfp3612, encoding a CCCH tandem zinc finger RNA-binding protein, results in defective hematopoiesis. Blood 114(12):2401-2410.

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Alternative Mouse Model for Asthma Reveals Novel Pathways

To understand some of the physiological factors initiating allergic asthma, researchers from NIEHS and the Children's Hospital of Pittsburgh compared two models of allergic sensitization in mice. They found that allergic sensitization through the airway, using ovalbumin (OVA) as an allergen, together with low-dose lipopolysaccharide (LPS) as an adjuvant, induced a very different immune response than the traditional mouse model of asthma, which relies on intraperitoneal injections of OVA together with the adjuvant, aluminum hydroxide.

Following these two types of sensitization, both groups of mice were challenged with aerosolized OVA. Although mucus production in the lung was similar in both groups, airway levels of eosinophils and T helper (Th)2 cell cytokines were significantly higher in mice sensitized through the peritoneum. However, these mice had low levels of airway neutrophils and did not develop allergen-induced airway hyperreactivity (AHR).

In contrast, mice sensitized through the airway displayed only modest Th2 responses, but had high levels of neutrophils and Th17 cells in the lung, and exhibited robust AHR. The pro-inflammatory Th17 cells released the cytokine IL-17 into the airway, which in turn led to neutrophil recruitment and AHR. Neutrophils, which are white blood cells associated with inflammation, are also increased in lungs of humans with severe asthma.

The findings in this paper suggest that Th17 and Th2 responses act synergistically to promote airway neutrophilia and AHR. Blocking neutrophil recruitment to the lung prevented AHR in the mice, suggesting that a similar approach might also be an effective therapy in humans.

Citation: Wilson RH, Whitehead GS, Nakano H, Free ME, Kolls JK, Cook DN. ( NIEHS 2009. Allergic sensitization through the airway primes Th17-dependent neutrophila and airway hyperresponsiveness. Am J Respir Crit Care Med 180(8):720-730.

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Calcium Sensing Protein is Specialized for Digital Signaling

Many cells utilize calcium as an important second messenger. Physiological receptor activation induces repetitive intracellular calcium release events, called calcium oscillations, a regenerative process that involves the release of calcium from the endoplasmic reticulum (ER).

Researchers at NIEHS demonstrate how these "digital" calcium release events are sufficient to cause two transmembrane ER proteins, STIM1 and STIM2, to aggregate and migrate toward the plasma membrane. The proximity of these STIM proteins to the plasma membrane is crucial for the activation of store-operated calcium entry (SOCE), a calcium entry process dependent on calcium channels made up of the Orai proteins. Importantly, a sustained oscillatory calcium response is dependent on SOCE to replenish ER calcium stores.

The group combined intracellular calcium measurements, using fluorescent calcium indicators, with total internal reflection fluorescence microscopy (TIRFM) to simultaneously monitor agonist-induced oscillatory calcium responses and the movement of STIM proteins, respectively. The researchers reported that STIM1, not STIM2, was necessary for activating SOCE and sustaining intracellular calcium oscillations.

The study also provides remarkable evidence that during calcium oscillations, the movement of STIM1 proteins is coordinated with transient calcium release events. By this manner, the transient drops in ER calcium stores are digitally encoded by STIM1 and may activate SOCE with a similar time course. Thus, under physiological conditions, this arrangement provides a clearly defined and unambiguous signaling system, translating a digital calcium release signal into calcium influx that can signal to downstream effectors.

Citation: Bird GS, Hwang SY, Smyth JT, Fukushima M, Boyles RR, Putney JW Jr. ( NIEHS 2009. STIM1 is a calcium sensor specialized for digital signaling. Curr Biol 19(20:1724-1729.

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Polymerase Stalling Controls Inflammatory Gene Expression

RNA Polymerase II stalling ensures a timely and coordinated activation of proinflammatory gene expression in immune cells. Using immune cells from both mice and Drosophila, researchers from NIEHS and Weil Medical College of Cornell University studied the kinetics of cytokine gene regulation in response to microbial stimulus. They determined that the key difference between rapid responding genes and slower activated genes is the step in the transcription cycle at which they are regulated.

Karen Adelman, Ph.D., lead author on the study and principal investigator in the Laboratory of Molecular Carcinogenesis, showed that in quickly activated genes, such as TNF alpha, RNA Polymerase II initiates transcription, but stalls near the promoter region, held back by the negative elongation factor (NELF). Upon macrophage stimulation, NELF is shed from the polymerase complex and the polymerase is released into the gene, thus promoting rapid synthesis of cytokine gene products. In contrast, both polymerase and NELF are absent in promoter regions of slow response genes such as IP-10. The investigators determined that recruitment of polymerase is the rate-limiting factor for transcription of these genes.

Adelman's group used chromatin immunoprecipitation (ChIP) and permanganate probing to establish the molecular mechanisms involved in polymerase stalling. The group concluded that there is a high degree of evolutionary conservation of the transcriptional regulatory mechanisms governing inflammatory gene expression from Drosophila to mammals.

Citation: Adelman K, Kennedy MA, Nechaev S, Gilchrist DA, Muse GW, Chinenov Y, Rogatsky I. ( NIEHS 2009. Immediate mediators of the inflammatory response are poised for gene activation through RNA polymerase II stalling. Proc Natl Acad Sci USA 106(43):18207-18212.

(Laura Hall is a biologist in the NIEHS Laboratory of Pharmacology currently on detail as a writer for the Environmental Factor. Thaddeus Schug, Ph.D., is a postdoctoral research fellow in the NIEHS Laboratory of Signal Transduction.)

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