Star-PAP control of BIK expression and apoptosis is regulated by nuclear PIPKIα and PKCδ signaling.

BIK protein is an initiator of mitochondrial apoptosis, and BIK expression is induced by proapoptotic signals, including DNA damage. Here, we demonstrate that 3' end processing and expression of BIK mRNA are controlled by the nuclear PI4,5P(2)-regulated poly(A) polymerase Star-PAP downstream of DNA damage. Nuclear PKCδ is a key mediator of apoptosis, and DNA damage stimulates PKCδ association with the Star-PAP complex where PKCδ is required for Star-PAP-dependent BIK expression. PKCδ binds the PI4,5P(2)-generating enzyme PIPKIα, which is essential for PKCδ interaction with the Star-PAP complex, and PKCδ activity is directly stimulated by PI4,5P(2). Features in the BIK 3' UTR uniquely define Star-PAP specificity and may block canonical PAP activity toward BIK mRNA. This reveals a nuclear phosphoinositide signaling nexus where PIPKIα, PI4,5P(2), and PKCδ regulate Star-PAP control of BIK expression and induction of apoptosis. This pathway is distinct from the Star-PAP-mediated oxidative stress pathway indicating signal-specific regulation of mRNA 3' end processing.

Potential Airborne Asbestos Exposure and Risk Associated with the Historical Use of Cosmetic Talcum Powder Products.

Over time, concerns have been raised regarding the potential for human exposure and risk from asbestos in cosmetic-talc-containing consumer products. In 1985, the U.S. Food and Drug Administration (FDA) conducted a risk assessment evaluating the potential inhalation asbestos exposure associated with the cosmetic talc consumer use scenario of powdering an infant during diapering, and found that risks were below levels associated with background asbestos exposures and risk. However, given the scope and age of the FDA's assessment, it was unknown whether the agency's conclusions remained relevant to current risk assessment practices, talc application scenarios, and exposure data. This analysis updates the previous FDA assessment by incorporating the current published exposure literature associated with consumer use of talcum powder and using the current U.S. Environmental Protection Agency's (EPA) nonoccupational asbestos risk assessment approach to estimate potential cumulative asbestos exposure and risk for four use scenarios: (1) infant exposure during diapering; (2) adult exposure from infant diapering; (3) adult exposure from face powdering; and (4) adult exposure from body powdering. The estimated range of cumulative asbestos exposure potential for all scenarios (assuming an asbestos content of 0.1%) ranged from 0.0000021 to 0.0096 f/cc-yr and resulted in risk estimates that were within or below EPA's acceptable target risk levels. Consistent with the original FDA findings, exposure and corresponding health risk in this range were orders of magnitude below upper-bound estimates of cumulative asbestos exposure and risk at ambient levels, which have not been associated with increased incidence of asbestos-related disease.

Historical evolution of regulatory standards for occupational and consumer exposures to industrial talc.

Talc has been used historically in a wide range of industrial applications and consumer products. The composition and purity of talc used for industrial purposes can vary greatly depending on the source and may contain asbestos minerals. The developing science associated with the health risks of asbestos had an effect on the talc industry throughout the 20th century. This review presents a detailed analysis of the evolution of regulatory standards impacting the use of industrial talc in the U.S. from the early 20th century through the 1990s. While it was recognized by the 1930s that airborne exposures to talc dust at high concentrations could cause lung disease, it was not until later that concerns were raised about the health risks associated with potential occupational exposures to asbestos from industrial talc. Regulatory agencies adopted occupational standards for industrial talc in the early 1970s, but the terminology used to define and characterize talc and other associated minerals varied between agencies. In addition, the complex and varying mineralogy of industrial talc led to inconsistent and imprecise interpretation of studies concerning health risk and occupational health standards among individual agencies.

History of knowledge and evolution of occupational health and regulatory aspects of asbestos exposure science: 1900-1975.

The understanding by industrial hygienists of the hazards of asbestos and appropriate ways to characterize and control exposure has evolved over the years. Here, a detailed analysis of the evolution of industrial hygiene practices regarding asbestos and its health risks, from the early 1900s until the advent of the national occupational health and safety regulatory structure currently in place in the US (early-to-mid 1970s) is presented. While industrial hygienists recognized in the early 1900s that chronic and high-level exposures to airborne concentrations of asbestos could pose a serious health hazard, it was not until the mid-1950s that the carcinogenic nature of asbestos began to be characterized and widespread concern followed. With the introduction of the membrane filter sampling method in the late 1960s and early 1970s, asbestos sampling and exposure assessment capabilities advanced to a degree which allowed industrial hygienists to more precisely characterize the exposure-response relationship. The ability of industrial hygienists, analytical chemists, toxicologists, and physicians to more accurately define this relationship was instrumental to the scientific community's ability to establish Occupational Exposure Levels (OELs) for asbestos. These early developments set the stage for decades of additional study on asbestos exposure potential and risk of disease. This was followed by the application of engineering controls and improved respiratory protection which, over the years, saved thousands of lives. This paper represents a state-of-the-art review of the knowledge of asbestos within the industrial hygiene community from about 1900 to 1975.

Asbestos fiber length and its relation to disease risk.

Differences in chemical and crystalline composition, fiber dimension, aerodynamic characteristics and biodurability are among the critical factors that define the toxicological and pathological consequences of asbestos exposure. Specifically, fiber dimension can impact whether the fiber is respired, whether and how deeply it is deposited in the lung, and how efficiently and rapidly it may be cleared. This paper provides a current, comprehensive evaluation of the weight of evidence regarding the relationship between asbestos fiber length and disease potency (for malignant and nonmalignant endpoints). In vitro studies, animal exposure studies and epidemiology data were reviewed. We found that the data reported over the last several decades consistently support the conclusions that exposure to fibers longer than 10 µm and perhaps 20 µm are required to significantly increase the risk of developing asbestos-related disease in humans and that there is very little, if any, risk associated with exposure to fibers shorter than 5 µm. Fiber length as a predictor of potency has been evaluated by several federal agencies in the U.S. and could significantly influence future regulatory decisions for elongated mineral particles (EMPs) and high-aspect ratio nanoparticles (HARNs).

A PtdIns4,5P2-regulated nuclear poly(A) polymerase controls expression of select mRNAs.

Phosphoinositides are a family of lipid signalling molecules that regulate many cellular functions in eukaryotes. Phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2), the central component in the phosphoinositide signalling circuitry, is generated primarily by type I phosphatidylinositol 4-phosphate 5-kinases (PIPKIalpha, PIPKIbeta and PIPKIgamma). In addition to functions in the cytosol, phosphoinositides are present in the nucleus, where they modulate several functions; however, the mechanism by which they directly regulate nuclear functions remains unknown. PIPKIs regulate cellular functions through interactions with protein partners, often PtdIns4,5P2 effectors, that target PIPKIs to discrete subcellular compartments, resulting in the spatial and temporal generation of PtdIns4,5P2 required for the regulation of specific signalling pathways. Therefore, to determine roles for nuclear PtdIns4,5P2 we set out to identify proteins that interacted with the nuclear PIPK, PIPKIalpha. Here we show that PIPKIalpha co-localizes at nuclear speckles and interacts with a newly identified non-canonical poly(A) polymerase, which we have termed Star-PAP (nuclear speckle targeted PIPKIalpha regulated-poly(A) polymerase) and that the activity of Star-PAP can be specifically regulated by PtdIns4,5P2. Star-PAP and PIPKIalpha function together in a complex to control the expression of select mRNAs, including the transcript encoding the key cytoprotective enzyme haem oxygenase-1 (refs 8, 9) and other oxidative stress response genes by regulating the 3'-end formation of their mRNAs. Taken together, the data demonstrate a model by which phosphoinositide signalling works in tandem with complement pathways to regulate the activity of Star-PAP and the subsequent biosynthesis of its target mRNA. The results reveal a mechanism for the integration of nuclear phosphoinositide signals and a method for regulating gene expression.

The role of genotoxicity in asbestos-induced mesothelioma: an explanation for the differences in carcinogenic potential among fiber types.

The mechanism(s) underlying asbestos toxicity associated with the pathogenesis of mesothelioma has been a challenge to unravel for more than 60 years. A significant amount of research has focused on the characteristics of different fiber types and their potential to induce mesothelioma. These mechanistic studies of fiber toxicity have proceeded along two lines: those demonstrating biochemical mechanisms by which fibers induce disease and those investigating human susceptibility. Most recent studies focused on in vitro genotoxic effects induced by asbestos as the mechanism responsible for asbestos-induced disease. Although asbestos exerts a genotoxic effect at certain concentrations in vitro, a positive response in these tests does not indicate that the chemical is likely to produce an increased risk of carcinogenesis in exposed human populations. Thus far, findings from studies on the effects of fiber type in mesothelial cells are seriously flawed by a lack of a dose response relationship. The common limitation of these in vitro experiments is the lack of attention paid to the complexities of the human anatomy, biochemistry and physiology, which make the observed effects in these experimental systems difficult to extrapolate to persons in the workplace. Mechanistic differences between carcinogenic and genotoxic processes indicate why tests for genotoxicity do not provide much insight regarding the ability to predict carcinogenic potential in workers exposed to asbestos doses in the post-Occupational Safety and Health Administration era. This review discusses the existing literature on asbestos-induced genotoxicity and explains why these studies may or may not likely help characterize the dose-response curve at low dose.

CKI isoforms α and ε regulate Star-PAP target messages by controlling Star-PAP poly(A) polymerase activity and phosphoinositide stimulation.

Star-PAP is a non-canonical, nuclear poly(A) polymerase (PAP) that is regulated by the lipid signaling molecule phosphatidylinositol 4,5 bisphosphate (PI4,5P(2)), and is required for the expression of a select set of mRNAs. It was previously reported that a PI4,5P(2) sensitive CKI isoform, CKIα associates with and phosphorylates Star-PAP in its catalytic domain. Here, we show that the oxidative stress-induced by tBHQ treatment stimulates the CKI mediated phosphorylation of Star-PAP, which is critical for both its polyadenylation activity and stimulation by PI4,5P(2). CKI activity was required for the expression and efficient 3'-end processing of its target mRNAs in vivo as well as the polyadenylation activity of Star-PAP in vitro. Specific CKI activity inhibitors (IC261 and CKI7) block in vivo Star-PAP activity, but the knockdown of CKIα did not equivalently inhibit the expression of Star-PAP targets. We show that in addition to CKIα, Star-PAP associates with another CKI isoform, CKIε in the Star-PAP complex that phosphorylates Star-PAP and complements the loss of CKIα. Knockdown of both CKI isoforms (α and ε) resulted in the loss of expression and the 3'-end processing of Star-PAP targets similar to the CKI activity inhibitors. Our results demonstrate that CKI isoforms α and ε modulate Star-PAP activity and regulates Star-PAP target messages.

Evaluation of tremolite asbestos exposures associated with the use of commercial products.

Tremolite is a noncommercial form of amphibole mineral that is present in some chrysotile, talc, and vermiculite deposits. Inhalation of asbestiform tremolite is suspected to have caused or contributed to an increased incidence of mesothelioma in certain mining settings; however, very little is known about the magnitude of tremolite exposure that occurred at these locations, and even less is known regarding tremolite exposures that might have occurred during consumer use of chrysotile, talc, and vermiculite containing products. The purpose of this analysis is to evaluate the exposure-response relationship for tremolite asbestos and mesothelioma in high exposure settings (mining) and to develop estimates of tremolite asbestos exposure for various product use scenarios. Our interpretation of the tremolite asbestos exposure metrics reported for the Thetford chrysotile mines and the Libby vermiculite deposits suggests a lowest-observed-adverse-effect level (LOAEL) for mesothelioma of 35-73 f/cc-year. Using measured and estimated airborne tremolite asbestos concentrations for simulated and actual product use, we conservatively estimated the following cumulative tremolite asbestos exposures: career auto mechanic: 0.028 f/cc-year; non-occupational use of joint compound: 0.0006 f/cc-year; non-occupational use of vermiculite-containing gardening products: 0.034 f/cc-year; home-owner removal of Zonolite insulation: 0.0002 f/cc-year. While the estimated consumer tremolite exposures are far below the tremolite LOAELs derived herein, this analysis examines only a few of the hundreds of chrysotile- and talc-containing products.

Recruitment of the Arp2/3 complex to vinculin: coupling membrane protrusion to matrix adhesion.

Cell migration involves many steps, including membrane protrusion and the development of new adhesions. Here we have investigated whether there is a link between actin polymerization and integrin engagement. In response to signals that trigger membrane protrusion, the actin-related protein (Arp)2/3 complex transiently binds to vinculin, an integrin-associated protein. The interaction is regulated, requiring phosphatidylinositol-4,5-bisphosphate and Rac1 activation, and is sufficient to recruit the Arp2/3 complex to new sites of integrin aggregation. Binding of the Arp2/3 complex to vinculin is direct and does not depend on the ability of vinculin to associate with actin. We have mapped the binding site for the Arp2/3 complex to the hinge region of vinculin, and a point mutation in this region selectively blocks binding to the Arp2/3 complex. Compared with WT vinculin, expression of this mutant in vinculin-null cells results in diminished lamellipodial protrusion and spreading on fibronectin. The recruitment of the Arp2/3 complex to vinculin may be one mechanism through which actin polymerization and membrane protrusion are coupled to integrin-mediated adhesion.

Protein kinase A-mediated CREB phosphorylation is an oxidant-induced survival pathway in alveolar type II cells.

Oxidant stress plays a role in the pathogenesis of pulmonary diseases, including fibrotic lung disease and cancer. We previously found that hydrogen peroxide (H2O2) initiates an increase in Ca2+/cAMP-response element binding protein (CREB) phosphorylation in C10 alveolar type II cells that requires activation of extracellular regulated kinases 1/2 (ERK1/2). Here, we investigated the role of crosstalk between protein kinase A (PKA) and epidermal growth factor receptor (EGFR) in oxidant-induced signaling to ERK1/2 and CREB in C10 cells. Application of H2O2 increased nuclear accumulation of PKA, and inhibition of PKA with H89 reduced oxidant-mediated phosphorylation of both CREB and ERK1/2. Single cell measurements of cAMP and redox status, using a FRET-based biosensor and a redox-sensitive GFP, respectively, indicated that H2O2 increases production of cAMP that correlates with redox state. Inhibition of EGFR activity decreased both H2O2-induced CREB phosphorylation and translocation of PKA to the nucleus, suggesting that crosstalk between PKA and EGFR underlies the oxidant-induced CREB response. Furthermore, knockdown of CREB expression using siRNA led to a decrease in bcl-2 and an increase in oxidant-induced apoptosis. Together these data reveal a novel role for crosstalk between PKA, ERK1/2 and CREB that mediates cell survival during oxidant stress.

Airborne asbestos take-home exposures during handling of chrysotile-contaminated clothing following simulated full shift workplace exposures.

The potential for para-occupational, domestic, or take-home exposures from asbestos-contaminated work clothing has been acknowledged for decades, but historically has not been quantitatively well characterized. A simulation study was performed to measure airborne chrysotile concentrations associated with laundering of contaminated clothing worn during a full shift work day. Work clothing fitted onto mannequins was exposed for 6.5 h to an airborne concentration of 11.4 f/cc (PCME) of chrysotile asbestos, and was subsequently handled and shaken. Mean 5-min and 15-min concentrations during active clothes handling and shake-out were 3.2 f/cc and 2.9 f/cc, respectively (PCME). Mean airborne PCME concentrations decreased by 55% 15 min after clothes handling ceased, and by 85% after 30 min. PCM concentrations during clothes handling were 11-47% greater than PCME concentrations. Consistent with previously published data, daily mean 8-h TWA airborne concentrations for clothes-handling activity were approximately 1.0% of workplace concentrations. Similarly, weekly 40-h TWAs for clothes handling were approximately 0.20% of workplace concentrations. Estimated take-home cumulative exposure estimates for weekly clothes handling over 25-year working durations were below 1 f/cc-year for handling work clothes contaminated in an occupational environment with full shift airborne chrysotile concentrations of up to 9 f/cc (8-h TWA).

Nuclear phosphoinositides: a signaling enigma wrapped in a compartmental conundrum.

While the presence of phosphoinositides in the nuclei of eukaryotes and the identity of the enzymes responsible for their metabolism have been known for some time, their functions in the nucleus are only now emerging. This is illustrated by the recent identification of effectors for nuclear phosphoinositides. Like the cytosolic phosphoinositide signaling pathway, nuclear phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) is at the center of the pathway and acts both as a messenger and as a precursor for many additional messengers. Here, recent advances in the understanding of nuclear phosphoinositide signaling and its functions are reviewed with an emphasis on PI4,5P(2) and its role in gene expression. The compartmentalization of nuclear phosphoinositide phosphates (PIP(n)) remains a mystery, but emerging evidence suggests that phosphoinositides occupy several functionally distinct compartments.

Asbestos-mediated CREB phosphorylation is regulated by protein kinase A and extracellular signal-regulated kinases 1/2.

Asbestos is a ubiquitous, naturally occurring fiber that has been linked to the development of malignant and fibrotic lung diseases. Asbestos exposure leads to apoptosis, followed by compensatory proliferation, yet many of the signaling cascades coupled to these outcomes are unclear. Because CREs (Ca(2+)/cAMP-response elements) are found in the promoters of many genes important for regulation of proliferation and apoptosis, CREB (CRE binding protein) is likely to play an important role in the development of asbestos-mediated lung injury. To explore this possibility, we tested the hypotheses that asbestos exposure leads to CREB phosphorylation in lung epithelial cells and that protein kinase A (PKA) and extracellular signal-regulated kinases 1/2 (ERK1/2) are central regulators of the CREB pathway. Persistent CREB phosphorylation was observed in lung sections from mice following inhalation of crocidolite asbestos. Exposure of C10 lung epithelial cells to crocidolite asbestos led to rapid CREB phosphorylation and apoptosis that was decreased by the inhibition of PKA or ERK1/2 using the specific inhibitors H89 and U0126, respectively. Furthermore, crocidolite asbestos selectively induced a sustained increase in MAP kinase phosphatase-1 mRNA and protein. Silencing CREB protein dramatically reduced asbestos-mediated ERK1/2 phosphorylation, yet significantly increased the number of cells undergoing asbestos-induced apoptosis. These data reveal a novel and selective role for CREB in asbestos-mediated signaling through pathways regulated by PKA and ERK1/2, further providing evidence that CREB is an important regulator of apoptosis in asbestos-induced responses of lung epithelial cells.

Oxidant-mediated cAMP response element binding protein activation: calcium regulation and role in apoptosis of lung epithelial cells.

Oxidant stress-mediated regulation of extracellular signal-regulated kinases (ERK1/2) is linked to pathologic outcomes in lung epithelium, yet a role for Ca2+ and Ca2+/cAMP-response element binding protein (CREB) in ERK1/2 signaling has not been defined. In this study, we tested the hypotheses that oxidants induce Ca2+-mediated phosphorylation of ERK and CREB, and that CREB is required for oxidant-induced proliferation and apoptosis. H2O2 initiated an influx of extracellular Ca2+ that was required for phosphorylation of both ERK and CREB in C10 lung epithelial cells. H2O2-mediated CREB phosphorylation was sensitive to MEK inhibition, suggesting that crosstalk between Ca2+, ERK, and CREB signaling pathways contributes to the oxidant-induced response. Reduction of CREB activity, using a dominant-negative CREB construct, inhibited c-fos steady-state mRNA levels, but unexpectedly enhanced bcl-2 steady-state mRNA levels after H2O2 exposure. Whereas inhibition of CREB activity had no detectable effect on H2O2 stimulation of cell cycle, loss of CREB activity significantly reduced the number of cells undergoing apoptosis. These data support a novel communication between Ca2+-ERK1/2 and CREB elicited by H2O2, and further provide evidence that CREB is an important regulator of apoptosis in oxidant-mediated responses of lung epithelial cells.

Excitation-transcription coupling in smooth muscle.

Calcium (Ca2+) signals affect virtually every biological process, including both contraction and gene transcription in smooth muscle. Ca2+-regulated gene transcription is known to be important for both physiological and pathological responses in smooth muscle. The aim of this review is to discuss the current understanding of gene transcription regulated by excitation through Ca2+ signalling using a comparison of the two most characterized Ca2+-regulated transcription factors in smooth muscle, Ca2+-cyclic AMP response element binding protein (CREB) and nuclear factor of activated T-cells (NFAT). Recent studies have shown commonalities and differences in the regulation of CREB and NFAT through both voltage- and non-voltage-gated Ca2+ channels that lead to expression of smooth muscle cell specific differentiation markers as well as markers of proliferation. New insights into the regulation of specific genes through companion elements on the promoters of Ca2+-regulated genes have led to new models for transcriptional regulation by Ca2+ that are defined both by the source and duration of the Ca2+ signal and the composition of enhancer elements found within the regulatory regions of specific genes. Thus the combination of signalling pathways elicited by particular Ca2+ signals affect selective promoter elements that are key to the ultimate pattern of gene transcription.

Ca2+ source-dependent transcription of CRE-containing genes in vascular smooth muscle.

Altered Ca2+ handling has immediate physiological and long-term genomic effects on vascular smooth muscle function. Previously we showed that Ca2+ entry through voltage-dependent Ca2+ channels (VDCCs) or store-operated Ca2+ channels (SOCCs) results in phosphorylation of the Ca2+/cAMP response element (CRE)-binding protein in cerebral arteries. Here, oligonucleotide array analysis was used to determine gene transcription profiles resulting from these two Ca2+ entry pathways in human cerebrovascular smooth muscle cell cultures. Results were confirmed and expanded using quantitative RT-PCR, Western blot, and immunofluorescence. A distinct, yet overlapping, set of CRE-regulated genes was induced by VDCC activation using K+ membrane depolarization vs. SOCC activation by thapsigargin (TG). Membrane depolarization selectively induced a sustained increase in early growth response-1 (Egr-1) mRNA and protein, which were inhibited by the VDCC blocker nimodipine and the SOCC inhibitor 2-aminoethoxydiphenylborate (2-APB). TG selectively induced a sustained increase in MAPK phosphatase-1 (MKP-1) mRNA and protein, and these effects were decreased by 2-APB, but not by nimodipine. The physiological agonist ANG II also stimulated expression of Egr-1 and MKP-1. Coadministration of 2-APB prevented expression of Egr-1 and MKP-1, whereas nimodipine blocked only Egr-1 expression. TG and ANG II induced phosphorylation of ERK, which was sensitive to 2-APB and was selectively required for CRE-binding protein phosphorylation. Our findings thus indicate that Ca2+ entry through VDCCs and store-operated Ca2+ entry can differentially regulate CRE-containing genes in vascular smooth muscle and also imply that agonist-induced signals involved in modulation of gene transcription can be controlled by multiple sources of Ca2+.