Sensitivity and activation of endoplasmic reticulum stress response and apoptosis in the perinatal sheep heart.

Although the unfolded protein response (UPR) contributes to survival by removing misfolded proteins, endoplasmic reticulum (ER) stress also activates proapoptotic pathways. Changed sensitivity to normal developmental stimuli may underlie observed cardiomyocyte apoptosis in the healthy perinatal heart. We determined in vitro sensitivity to thapsigargin in sheep cardiomyocytes from four perinatal ages. In utero cardiac activation of ER stress and apoptotic pathways was determined at these same ages. Thapsigargin-induced phosphorylation of eukaryotic initiation factor 2 (EIF2A) was decreased by 72% between 135 and 143 dGA (P = 0.0096) and remained low at 1 dPN (P = 0.0080). Conversely, thapsigargin-induced caspase cleavage was highest around the time of birth: cleaved caspase 3 was highest at 1 dPN (3.8-fold vs. 135 dGA, P = 0.0380; 7.8-fold vs. 5 dPN, P = 0.0118), cleaved caspase 7 and cleaved caspase 12 both increased between 135 and 143 dGA (25-fold and 6.9-fold respectively, both P < 0.0001) and remained elevated at 1 dPN. Induced apoptosis, measured by TdT-mediated dUTP nick-end labeling (TUNEL) assay, was highest around the time of birth (P < 0.0001). There were changes in myocardial ER stress pathway components in utero. Glucose (78 kDa)-regulated protein (GRP78) protein levels were high in the fetus and declined after birth (P < 0.0001). EIF2A phosphorylation was profoundly depressed at 1 dPN (vs. 143 dGA, P = 0.0113). In conclusion, there is dynamic regulation of ER proteostasis, ER stress, and apoptosis cascade in the perinatal heart. Apoptotic signaling is more readily activated in fetal cardiomyocytes near birth, leading to widespread caspase cleavage in the newborn heart. These pathways are important for the regulation of normal maturation in the healthy perinatal heart.NEW & NOTEWORTHY Cardiomyocyte apoptosis occurs even in the healthy, normally developing perinatal myocardium. As cardiomyocyte number is a critical contributor to heart health, the sensitivity of cardiomyocytes to endoplasmic reticulum stress leading to apoptosis is an important consideration. This study suggests that the heart has less robust protective mechanisms in response to endoplasmic reticulum stress immediately before and after birth, and that more cardiomyocyte death can be induced by stress in this period.

Absolute Quantification of Transcription Factors Reveals Principles of Gene Regulation in Erythropoiesis.

Dynamic cellular processes such as differentiation are driven by changes in the abundances of transcription factors (TFs). However, despite years of studies, our knowledge about the protein copy number of TFs in the nucleus is limited. Here, by determining the absolute abundances of 103 TFs and co-factors during the course of human erythropoiesis, we provide a dynamic and quantitative scale for TFs in the nucleus. Furthermore, we establish the first gene regulatory network of cell fate commitment that integrates temporal protein stoichiometry data with mRNA measurements. The model revealed quantitative imbalances in TFs' cross-antagonistic relationships that underlie lineage determination. Finally, we made the surprising discovery that, in the nucleus, co-repressors are dramatically more abundant than co-activators at the protein level, but not at the RNA level, with profound implications for understanding transcriptional regulation. These analyses provide a unique quantitative framework to understand transcriptional regulation of cell differentiation in a dynamic context.

Olfactory Transcriptional Analysis of Salmon Exposed to Mixtures of Chlorpyrifos and Malathion Reveal Novel Molecular Pathways of Neurobehavioral Injury.

Pacific salmon exposed to sublethal concentrations of organophosphate pesticides (OP) have impaired olfactory function that can lead to loss of behaviors that are essential for survival. These exposures often involve mixtures and can occur at levels below those which inhibit acetylcholinesterase (AChE). In this study, juvenile Coho salmon were exposed for 24 h to either 0.1, 0.5, or 2.5 ppb chlorpyrifos (CPF), 2, 10, or 50 ppb malathion (MAL), or binary mixtures of 0.1 CPF:2 ppb MAL, 0.5 CPF:10 ppb MAL, or 2.5 CPF:10 ppb MAL to mimic single and binary environmental exposures. Microarray analysis of olfactory rosettes from pesticide-exposed salmon revealed differentially expressed genes involved in nervous system function and signaling, aryl hydrocarbon receptor signaling, xenobiotic metabolism, and mitochondrial dysfunction. Coho exposed to OP mixtures exhibited a more pronounced loss in detection of a predatory olfactory cue relative to those exposed to single compounds, whereas respirometry experiments demonstrated that exposure to OPs, individually and in mixtures, reduced maximum respiratory capacity of olfactory rosette mitochondria. The observed molecular, biochemical, and behavioral effects occurred largely in the absence of effects on brain AChE. In summary, our results provide new insights associated with the sublethal neurotoxic effects of OP mixtures relevant to environmental exposures involving molecular and cellular pathways of injury to the salmon olfactory system that underlie neurobehavioral injury.

Effects of chronic hypoxia on cardiac function measured by pressure-volume catheter in fetal chickens.

Hypoxia is a common component of many developmental insults and has been studied in early-stage chicken development. However, its impact on cardiac function and arterial-ventricular coupling in late-stage chickens is relatively unknown. To test the hypothesis that hypoxic incubation would reduce baseline cardiac function but protect the heart during acute hypoxia in late-stage chickens, white Leghorn eggs were incubated at 21% O2 or 15% O2. At 90% of incubation (19 days), hypoxic incubation caused growth restriction (-20%) and increased the LV-to-body ratio (+41%). Left ventricular (LV) pressure-volume loops were measured in anesthetized chickens in normoxia and acute hypoxia (10% O2). Hypoxic incubation lowered the maximal rate of pressure generation (ΔP/ΔtMax; -22%) and output (-57%), whereas increasing end-systolic elastance (ELV; +31%) and arterial elastance (EA; +122%) at similar heart rates to normoxic incubation. Both hypoxic incubation and acute hypoxia lengthened the half-time of relaxation (τ; +24%). Acute hypoxia reduced heart rate (-8%) and increased end-diastolic pressure (+35%). Hearts were collected for mRNA analysis. Hypoxic incubation was marked by decreased mRNA expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase 2, Na(+)/Ca(2+) exchanger 1, phospholamban, and ryanodine receptor. In summary, hypoxic incubation reduces LV function in the late-stage chicken by slowing pressure generation and relaxation, which may be driven by altered intracellular excitation-contraction coupling. Cardiac efficiency is greatly reduced after hypoxic incubation. In both incubation groups acute hypoxia reduced diastolic function.

Brief exposure to copper induces apoptosis and alters mediators of olfactory signal transduction in coho salmon.

Pacific salmon are particularly susceptible to copper (Cu)-induced olfactory injuries that can ultimately inhibit neurobehaviors critical to survival. However, the molecular mechanisms underlying Cu-mediated olfactory impairment remain poorly understood. In the present study, we conducted a short-term Cu exposure at levels relevant to urban runoff (5, 25 and 50 ppb) , and investigated the roles of impaired olfactory signal transduction and induced apoptosis as underlying mechanisms of olfactory injury. Increased cell death in the olfactory epithelium was evident in coho receiving 4h exposures to 25 and 50 ppb Cu. Expression of olfactory marker protein (omp), a marker of mature olfactory sensory neurons, also decreased at 50 ppb Cu. Immunohistochemical analysis of coho olfactory epithelium demonstrated a loss of type 3 adenylate cyclase (ACIII) in the apical olfactory epithelium cilia at all levels of Cu exposure, suggesting an inhibitory effect of Cu in olfactory signaling. Accompanying the loss of ACIII in Cu-exposed coho were reduced intracellular cyclic guanosine monophosphate (cGMP) levels in the olfactory rosettes. Collectively, these results support a linkage among the initial steps of olfactory signaling in Cu-induced salmon olfactory injury, and suggesting that monitoring olfactory cGMP levels may aid in the assessment of salmon olfactory injury.

Cloning, expression and analysis of the olfactory glutathione S-transferases in coho salmon.

The glutathione S-transferases (GSTs) provide cellular protection by detoxifying xenobiotics, maintaining redox status, and modulating secondary messengers, all of which are critical to maintaining olfaction in salmonids. Here, we characterized the major coho salmon olfactory GSTs (OlfGSTs), namely omega, pi, and rho subclasses. OlfGST omega contained an open reading frame of 720bp and encoded a protein of 239 amino acids. OlfGST pi and OlfGST rho contained open reading frames of 627 and 681nt, respectively, and encoded proteins of 208 and 226 amino acids. Whole-protein mass spectrometry yielded molecular weights of 29,950, 23,354, and 26,655Da, respectively, for the GST omega, pi, and rho subunits. Homology modeling using four protein-structure prediction algorithms suggest that the active sites in all three OlfGST isoforms resembled counterparts in other species. The olfactory GSTs conjugated prototypical GST substrates, but only OlfGST rho catalyzed the demethylation of the pesticide methyl parathion. OlfGST pi and rho exhibited thiol oxidoreductase activity toward 2-hydroxyethyl disulfide (2-HEDS) and conjugated 4-hydroxynonenal (HNE), a toxic aldehyde with neurodegenerative properties. The kinetic parameters for OlfGST pi conjugation of HNE were K(M)=0.16 ± 0.06mM and V(max)=0.5 ± 0.1µmolmin⁻¹mg⁻¹, whereas OlfGST rho was more efficient at catalyzing HNE conjugation (K(M)=0.022 ± 0.008 mM and V(max)=0.47 ± 0.05µmolmin⁻¹mg⁻¹). Our findings indicate that the peripheral olfactory system of coho expresses GST isoforms that detoxify certain electrophiles and pesticides and that help maintain redox status and signal transduction.

Characterization of phospholipid hydroperoxide glutathione metabolizing peroxidase (gpx4) isoforms in Coho salmon olfactory and liver tissues and their modulation by cadmium.

Exposure to environmental contaminants, including various pesticides and trace metals, can disrupt critical olfactory-driven behaviors of fish such as homing to natal streams, mate selection, and an ability to detect predators and prey. These neurobehavioral injuries have been linked to reduced survival and population declines. Despite the importance of maintaining proper olfactory signaling processes in the presence of chemical exposures, little is known regarding chemical detoxification in the salmon olfactory system, and in particular, the antioxidant defenses that maintain olfactory function. An understudied, yet critical component of cellular antioxidant defense is phospholipid hydroperoxide glutathione peroxidase (PHGPx/GPx4), an isoform within the family of selenium-dependent glutathione peroxidase (GPx) enzymes that can directly reduce lipid peroxides and other membrane-bound complex hydroperoxides. In this study, we cloned two gpx4 isoforms (gpx4a and gpx4b) from Coho salmon olfactory tissues and compared their modulation in olfactory and liver tissues by cadmium, an environmental pollutant and olfactory toxicant that cause oxidative damage as a mechanism of toxicity. Amino acid sequence comparisons of the two gpx4 isoforms shared 71% identity, and also relatively high sequence identities when compared with other fish GPx4 isoforms. Sequence comparisons with human GPx4 indicated conservation of three important active sites at selenocysteine (U46), glutamine (Q81), and tryptophan (W136), suggesting similar catalytic activity between fish and mammalian GPx4 isoforms. Tissue profiling confirmed the expression of gpx4a and gpx4b in all ten Coho tissues examined. The expression of gpx4 mRNAs in the Coho olfactory system was accompanied by comparably high initial rates of GPx4 enzymatic activity in mitochondrial and cytosolic fractions. Exposure to low (3.7 ppb) and high (347 ppb) environmental Cd concentrations for 24-48 h significantly decreased gpx4a expression in Coho olfactory rosettes, whereas olfactory gpx4b mRNA expression was not modulated by exposures at these concentrations. In summary, Coho salmon express two paralogs of gpx4, a key enzyme in the maintenance of signal transduction processes that protect against cellular oxidative damage. The Cd-associated downregulation of salmon olfactory gpx4a expression in particular, may be associated with the loss of olfactory signal transduction that accompanies metal-associated loss of olfaction in salmonids.

Effect of cadmium on glutathione S-transferase and metallothionein gene expression in coho salmon liver, gill and olfactory tissues.

The glutathione S-transferases (GSTs) are a multifunctional family of phase II enzymes that detoxify a variety of environmental chemicals, reactive intermediates, and secondary products of oxidative damage. GST mRNA expression and catalytic activity have been used as biomarkers of exposure to environmental chemicals. However, factors such as species differences in induction, partial analyses of multiple GST isoforms, and lack of understanding of fish GST gene regulation, have confounded the use of GSTs as markers of pollutant exposure. In the present study, we examined the effect of exposure to cadmium (Cd), a prototypical environmental contaminant and inducer of mammalian GST, on GST mRNA expression in coho salmon (Oncorhynchus kisutch) liver, gill, and olfactory tissues. GST expression data were compared to those for metallothionein (MT), a prototypical biomarker of metal exposure. Data mining of genomic databases led to the development of quantitative real-time PCR (qPCR) assays for salmon GST isoforms encompassing 9 subfamilies, including alpha, mu, pi, theta, omega, kappa, rho, zeta and microsomal GST. In vivo acute (8-48 h) exposures to low (3.7 ppb) and high (347 ppb) levels of Cd relevant to environmental scenarios elicited a variety of transient, albeit minor changes (<2.5-fold) in tissue GST profiles, including some reductions in GST mRNA expression. In general, olfactory GSTs were the earliest to respond to cadmium, whereas, more pronounced effects in olfactory and gill GST expression were observed at 48 h relative to earlier time points. Although evaluation of GSTs reflected a cadmium-associated oxidative stress response, there was no clear GST isoform in any tissue that could serve as a reliable biomarker of acute cadmium exposure. By contrast, metallothionein (MT) mRNA was consistently and markedly induced in all three tissues by cadmium, and among the tissues examined, olfactory MT was the most sensitive marker of cadmium exposures. In summary, coho salmon exhibit a complex GST tissue profile consisting of at least 9 isoforms, all of which are present in the peripheral olfactory system. Short-term exposure to environmental levels of Cd causes transient changes in salmon GST consistent with oxidative stress, and in some cases, includes a loss of GST. In a biomarker context, however, monitoring of tissue MT mRNA expression, especially in the peripheral olfactory system, may be of greater utility for assessing short-term environmental exposures to cadmium.

Chromatin-associated HMG-17 is a major regulator of homeodomain transcription factor activity modulated by Wnt/beta-catenin signaling.

Homeodomain (HD) transcriptional activities are tightly regulated during embryogenesis and require protein interactions for their spatial and temporal activation. The chromatin-associated high mobility group protein (HMG-17) is associated with transcriptionally active chromatin, however its role in regulating gene expression is unclear. This report reveals a unique strategy in which, HMG-17 acts as a molecular switch regulating HD transcriptional activity. The switch utilizes the Wnt/beta-catenin signaling pathway and adds to the diverse functions of beta-catenin. A high-affinity HMG-17 interaction with the PITX2 HD protein inhibits PITX2 DNA-binding activity. The HMG-17/PITX2 inactive complex is concentrated to specific nuclear regions primed for active transcription. beta-Catenin forms a ternary complex with PITX2/HMG-17 to switch it from a repressor to an activator complex. Without beta-catenin, HMG-17 can physically remove PITX2 from DNA to inhibit its transcriptional activity. The PITX2/HMG-17 regulatory complex acts independently of promoter targets and is a general mechanism for the control of HD transcriptional activity. HMG-17 is developmentally regulated and its unique role during embryogenesis is revealed by the early embryonic lethality of HMG-17 homozygous mice. This mechanism provides a new role for canonical Wnt/beta-catenin signaling in regulating HD transcriptional activity during development using HMG-17 as a molecular switch.

Functional interactions between Dlx2 and lymphoid enhancer factor regulate Msx2.

Dlx2, Lymphoid Enhancer Factor (Lef-1) and Msx2 transcription factors are required for several developmental processes. To understand the control of gene expression by these factors, chromatin immunoprecipitation (ChIP) assays identified Msx2 as a downstream target of Dlx2 and Lef-1. Dlx2 activates the Msx2 promoter in several cell lines and binds DNA as a monomer and dimer. A Lef-1 beta-catenin-dependent isoform minimally activates the Msx2 promoter and a Lef-1 beta-catenin-independent isoform is inactive, however co-expression of Dlx2 and both Lef-1 isoforms synergistically activate the Msx2 promoter. Co-immunoprecipitation and protein pull-down experiments demonstrate Lef-1 physically interacts with Dlx2. Deletion analyses of the Lef-1 protein reveal specific regions required for synergism with Dlx2. The Lef-1 beta-catenin binding domain (betaDB) is not required for its interaction with Dlx2. Msx2 can auto-regulate its promoter and repress Dlx2 activation. Msx2 repression of Dlx2 activation is dose-specific and both bind a common DNA-binding element. These transcriptional mechanisms correlate with the temporal and spatial expression of these factors and may provide a mechanism for the control of several developmental processes. We demonstrate new transcriptional activities for Dlx2, Msx2 and Lef-1 through protein interactions and identification of downstream targets.

Protein kinase C phosphorylation modulates N- and C-terminal regulatory activities of the PITX2 homeodomain protein.

PKC phosphorylation regulates PITX2 DNA binding and transcriptional activity. Mutation of individual PKC sites demonstrates the functional regulation of PITX2 through phosphorylation. Immunoprecipitation of PITX2 and a PITX2 PKC mutant protein reveal specific in vivo phosphorylation by PKC in transfected cells. The transcriptional activity of PITX2 is negatively regulated by N-terminal phosphorylation and positively regulated by C-terminal phosphorylation. We demonstrate a mechanism of increased PITX2 transcriptional activation through protein interactions facilitated by phosphorylation of the PITX2 C-terminal tail. Phosphorylation of the PITX2 C terminus enhances the interaction with cellular factors. In corroboration with the PITX2 PKC functional studies, a newly identified C-terminal PITX2 mutation associated with Axenfeld-Rieger syndrome (ARS) demonstrates reduced phosphorylation. This mutation (PITX2 DeltaT1261) creates a frameshift mutation in codon 227 resulting in 11 novel amino acids downstream followed by premature truncation of the protein. Three PKC sites in the C-terminal tail and OAR domain are deleted, which results in decreased transcriptional activation. PITX2 DeltaT1261 is unable to interact with a cellular factor to synergistically activate transcription and demonstrates the first link of ARS with defective PITX2 protein interactions. Gene expression profiling of homozygous Pitx2 mutant mouse tissue reveals decreased Dlx2 expression as a potential molecular basis for developmental defects associated with ARS patients. Overall, phosphorylation imparts another level of regulation to the activity of the PITX2 homeodomain protein during development.

PITX2, beta-catenin and LEF-1 interact to synergistically regulate the LEF-1 promoter.

PITX2, beta-catenin and lymphoid enhancer factor (LEF-1) are required for the inductive formation of several epithelial-derived organs, including teeth. Lef-1 is expressed in the dental epithelium after Pitx2, and both factors have overlapping expression patterns in the tooth bud and cap stages. Our analysis of Pitx2-/- mutant mice showed reduced Lef-1 expression in facial tissues by RT-PCR and quantitative RT-PCR. Consistent with these results we show that the human 2.5 kb LEF-1 promoter is activated by PITX2. Furthermore, the LEF-1 promoter is differentially activated by PITX2 isoforms, which are co-expressed in dental epithelium. The 2.5 kb LEF-1 promoter contains two regions that act to inhibit its transcription in concert with PITX2. The proximal region contains a Wnt-responsive element (WRE) that attenuates PITX2 activation. LEF-1 cannot autoregulate LEF-1 expression; however co-transfection of PITX2 and LEF-1 result in a synergistic activation of the 2.5 kb LEF-1 promoter. LEF-1 specifically interacts with the PITX2 C-terminal tail. Deletion of a distal 800 bp segment of the LEF-1 promoter resulted in enhanced PITX2 activation, and increased synergistic activation in the presence of LEF-1. Furthermore, beta-catenin in combination with PITX2 synergistically activates the LEF-1 promoter and this activation is independent of the Wnt-responsive element. beta-catenin directly interacts with PITX2 to synergistically regulate LEF-1 expression. We show a new mechanism where LEF-1 expression is regulated through PITX2, LEF-1 and beta-catenin direct physical interactions. LEF-1 and beta-catenin interactions with PITX2 provide new mechanisms for the regulation of PITX2 transcriptional activity.

PITX2 isoform-specific regulation of atrial natriuretic factor expression: synergism and repression with Nkx2.5.

PITX2 and Nkx2.5 are two of the earliest known transcriptional markers of vertebrate heart development. Pitx2-/- mice present with severe cardiac malformations and embryonic lethality, demonstrating a role for PITX2 in heart development. However, little is known about the downstream targets of PITX2 in cardiogenesis. We report here that the atrial natriuretic factor (ANF) promoter is a target of PITX2. PITX2A, PITX2B, and PITX2C isoforms differentially activate the ANF promoter. However, only PITX2C can synergistically activate the ANF promoter in the presence of Nkx2.5. We further demonstrate that the procollagen lysyl hydroxylase (PLOD1) promoter is regulated by Nkx2.5. Mechanistically, PITX2C and Nkx2.5 synergistically regulate ANF and PLOD1 expression through binding to their respective DNA elements. Surprisingly, PITX2A activation of the ANF and PLOD1 promoters is repressed by co-transfection of Nkx2.5 in the C3H10T1/2 embryonic fibroblast cell line. Pitx2a and Pitx2c are endogenously expressed in C3H10T1/2 cells, and these cells express factors that differentially regulate PITX2 isoform activities. We provide a new mechanism for the regulation of heart development by PITX2 isoforms through the regulation of ANF and PLOD1 gene expression and Nkx2.5 transcriptional activity.

A molecular basis for differential developmental anomalies in Axenfeld-Rieger syndrome.

Pitx2, a bicoid-like homeodomain transcription factor and Dlx2 are two transcriptional markers observed during early tooth development. PITX2 binds to bicoid and bicoid-like elements in the Dlx2 promoter and activates this promoter 30-fold in Chinese hamster ovary cells. Mutations in PITX2 associated with Axenfeld-Rieger syndrome (ARS) provided the first link of this homeodomain transcription factor to tooth development. We are investigating the molecular basis of developmental anomalies associated with human PITX2 mutations. A phenotypically less severe ARS mutant (without tooth anomalies), PITX2 R84W, has a similar DNA binding specificity compared to wild-type PITX2 and transactivates the Dlx2 promoter. This mutation is associated with iris hypoplasia (IH); in contrast a Rieger syndrome mutation, PITX2 T68P, which presents clinically with the full spectrum of developmental anomalies (including tooth anomalies), is unable to transactivate the Dlx2 promoter. Since Dlx2 expression is required for tooth and craniofacial development the lack of tooth anomalies in the patient with IH may be due to the residual activity of this mutant in activating the Dlx2 promoter. We demonstrate that PITX2 phosphorylation increases PITX2 and PITX2 R84W DNA binding. The PITX2 T68P ARS mutation occurs at a protein kinase C phosphorylation site in the homeodomain. Surprisingly, phosphorylation of PITX2 T68P is increased compared to wild-type PITX2 but has little effect on its DNA binding activity. Altogether these data suggest a molecular mechanism for tooth development involving Dlx2 gene expression in ARS patients.

Differential regulation of gene expression by PITX2 isoforms.

Three major PITX2 isoforms are differentially expressed in human, mice, zebrafish, chick, and frog tissues. To demonstrate differential regulation of gene expression by these isoforms we used three different promoters and three cell lines. Transient transfection of Chinese hamster ovary, HeLa, and LS-8 cell lines revealed differences in PITX2A and PITX2C activation of the PLOD1 and Dlx2 promoters, however, PITX2B is inactive. In contrast, PITX2B actives the pituitary-specific Prolactin promoter at higher levels than either PITX2A or PITX2C. Interestingly, co-transfection of either PITX2A or PITX2C with PITX2B results in a synergistic activation of the PLOD1 and Dlx2 promoters. Furthermore, PITX2 isoforms have different transcriptional activity dependent upon the cells used for transfection analysis. We have isolated a fourth PITX2 isoform (PITX2D) expressed only in humans, which acts to suppress the transcriptional activity of the other PITX2 isoforms. Electrophoretic mobility shift assays and glutathione S-transferase pull-down experiments demonstrated that all isoforms interact with PITX2D and that PITX2B forms heterodimeric complexes with PITX2A and PITX2C. Our research provides a molecular basis for differential gene regulation through the expression of PITX2 isoforms. PITX2 isoform activities are both promoter- and cell-specific, and our data reveal new mechanisms for PITX2-regulated gene expression.