Distamycin A Inhibits HMGA1-Binding to the P-Selectin Promoter and Attenuates Lung and Liver Inflammation during Murine Endotoxemia

Background: The architectural transcription factor High Mobility Group-A1 (HMGA1) binds to the minor groove of AT-rich DNA and forms transcription factor complexes (“enhanceosomes”) that upregulate expression of select genes within the inflammatory cascade during critical illness syndromes such as acute lung injury (ALI). AT-rich regions of DNA surround transcription factor binding sites in genes critical for the inflammatory response. Minor groove binding drugs (MGBs), such as Distamycin A (Dist A), interfere with AT-rich region DNA binding in a sequence and conformation-specific manner, and HMGA1 is one of the few transcription factors whose binding is inhibited by MGBs. Objectives: To determine whether MGBs exert beneficial effects during endotoxemia through attenuating tissue inflammation via interfering with HMGA1-DNA binding and modulating expression of adhesion molecules. Methodology/Principal Findings: Administration of Dist A significantly decreased lung and liver inflammation during murine endotoxemia. In intravital microscopy studies, Dist A attenuated neutrophil-endothelial interactions in vivo following an inflammatory stimulus. Endotoxin induction of P-selectin expression in lung and liver tissue and promoter activity in endothelial cells was significantly reduced by Dist A, while E-selectin induction was not significantly affected. Moreover, Dist A disrupted formation of an inducible complex containing NF-κB that binds an AT-rich region of the P-selectin promoter. Transfection studies demonstrated a critical role for HMGA1 in facilitating cytokine and NF-κB induction of P-selectin promoter activity, and Dist A inhibited binding of HMGA1 to this AT-rich region of the P-selectin promoter in vivo. Conclusions/Significance: We describe a novel targeted approach in modulating lung and liver inflammation in vivo during murine endotoxemia through decreasing binding of HMGA1 to a distinct AT-rich region of the P-selectin promoter. These studies highlight the ability of MGBs to function as molecular tools for dissecting transcriptional mechanisms in vivo and suggest alternative treatment approaches for critical illness

Misinformed Consent: Upholding the Constitutionality of South Dakota\u27s Suicide Advisory in \u3cem\u3ePlanned Parenthood Minnesota, North Dakota, South Dakota v. Rounds\u3c/em\u3e

On July 24, 2012, in Planned Parenthood Minnesota, North Dakota, South Dakota v. Rounds, the U.S. Court of Appeals for the Eighth Circuit, sitting en banc, considered the constitutionality of a suicide advisory portion of a South Dakota statute that required informed consent for abortions. The Eight Circuit found the advisory was constitutional because the information disclosures required by the statute were truthful, nonmisleading, and relevant to the patient’s decision to have an abortion. The court relied in part on the Supreme Court’s 2007 decision Gonzales v. Carhart, which held that Congress has the authority to legislate in the abortion context, even in areas of medical uncertainty. The Eighth Circuit, however, misapplied the reasoning of Gonzales and dangerously minimized the standard for scientific evidence in informed consent laws

Statutory Prohibitions on Wrongful Birth Claims & Their Dangerous Effects on Parents

Wrongful birth claims are negligence actions brought on behalf of children born with disabilities or genetic disorders that were not properly diagnosed before the child’s birth. The plaintiffs, typically the parents of the afflicted child, argue that without the defendant’s negligence, the parents would have had the opportunity to prevent the child’s birth and subsequent condition by choosing to terminate the pregnancy. A number of states have responded to the growing prevalence of wrongful birth claims by enacting legislation that bars plaintiffs from bringing wrongful birth actions. These statutes, however, pose a threat to the parental rights of disabled or terminally ill children, as they diminish abortion rights and bar parents from recovering the enormous medical and emotional damages of giving birth to the afflicted child. States should not prohibit this cause of action and, instead, the merits of these claims should be decided through the court system

La distamicina A inhibe la unión de HMGA1 al promotor de la selectina P y atenúa la inflamación pulmonar y hepática durante la endotoxemia murina

Background The architectural transcription factor High Mobility Group-A1 (HMGA1) binds to the minor groove of AT-rich DNA and forms transcription factor complexes (“enhanceosomes”) that upregulate expression of select genes within the inflammatory cascade during critical illness syndromes such as acute lung injury (ALI). AT-rich regions of DNA surround transcription factor binding sites in genes critical for the inflammatory response. Minor groove binding drugs (MGBs), such as Distamycin A (Dist A), interfere with AT-rich region DNA binding in a sequence and conformation-specific manner, and HMGA1 is one of the few transcription factors whose binding is inhibited by MGBs. Objectives To determine whether MGBs exert beneficial effects during endotoxemia through attenuating tissue inflammation via interfering with HMGA1-DNA binding and modulating expression of adhesion molecules. Methodology/Principal Findings Administration of Dist A significantly decreased lung and liver inflammation during murine endotoxemia. In intravital microscopy studies, Dist A attenuated neutrophil-endothelial interactions in vivo following an inflammatory stimulus. Endotoxin induction of P-selectin expression in lung and liver tissue and promoter activity in endothelial cells was significantly reduced by Dist A, while E-selectin induction was not significantly affected. Moreover, Dist A disrupted formation of an inducible complex containing NF-?B that binds an AT-rich region of the P-selectin promoter. Transfection studies demonstrated a critical role for HMGA1 in facilitating cytokine and NF-?B induction of P-selectin promoter activity, and Dist A inhibited binding of HMGA1 to this AT-rich region of the P-selectin promoter in vivo. Conclusions/Significance We describe a novel targeted approach in modulating lung and liver inflammation in vivo during murine endotoxemia through decreasing binding of HMGA1 to a distinct AT-rich region of the P-selectin promoter. These studies highlight the ability of MGBs to function as molecular tools for dissecting transcriptional mechanisms in vivo and suggest alternative treatment approaches for critical illness

Novel Microcephalic Primordial Dwarfism Disorder Associated with Variants in the Centrosomal Protein Ninein

Microcephalic primordial dwarfism (MPD) is a rare, severe form of human growth failure in which growth restriction is evident in utero and continues into postnatal life. Single causative gene defects have been identified in a number of patients with MPD, and all involve genes fundamental to cellular processes including centrosome functions

Distamycin A decreases lung and liver P-selectin tissue staining.

<p><b>A.</b> Representative sections (200× magnification) of lung tissue harvested from C57BL/6 mice 24 h after treatment with Vehicle (Veh), LPS+Veh, or LPS+Dist A (Dist). Tissue was fixed, processed, and stained with a P-selectin antibody. Number of positively stained vessels was counted in sections from at least 5 mice in each treatment group. (Arrows demonstrate examples of positively stained vessels; *p<0.05 compared with Veh; **p<0.05 compared with LPS/Veh). <b>B.</b> Representative sections (200× magnification) of liver tissue harvested from mice 4 h after treatment with Vehicle (Veh), LPS+Vehicle (Veh) or LPS+Dist A (Dist). Tissue was fixed, processed, and stained with a P-selectin antibody. Using NIH Image Software, 5 fields (at 200× magnification) per liver section from mice from each treatment group were quantified, with brown pixels counted as positive staining. Results were expressed as % positively stained area per 200× field. (*p<0.05 compared with Veh; **p<0.05 compared with LPS/Veh).</p

HMGA1 binds to the P-selectin promoter and is critical for full induction of P-selectin promoter activity.

<p><b>A.</b> BAEC cells were transiently transfected with a P-selectin promoter-reporter construct with the addition of a blank expression vector, an expression vector for HMGA1, and/or expression vectors for NF-κB family members (p50/p65). Transfected cells were harvested and assessed for luciferase activity (normalized for β-galactosidase content). Results are expressed as fold-change in luciferase activity relative to transfection with the P-selectin promoter and a blank expression vector. <b>B.</b> BAEC cells were transiently transfected with a P-selectin promoter-reporter construct and increasing concentrations of a vector expressing a dominant-negative form of HMGA1 (DN-HMGA1). Transfected cells were stimulated with TNF-α, then harvested and assessed for luciferase activity (normalized for β-galactosidase content). Results are expressed for each transfection condition as fold-change in luciferase activity as a result of TNF-α stimulation. (*p<0.05 for 0.5 µg of DN-HMGA1 as compared with empty vector control; **p<0.05 for 1.0 µg of DN-HMGA1 as compared with empty vector control). This experiment was repeated three separate times, with each condition performed in triplicate. <b>C.</b> An electrophoretic mobility shift assay (EMSA) was performed using the HMGA1 peptide and a radiolabeled probe spanning the AT-rich region of the P-selectin promoter (basepairs −542 to −521) without (Lane 2) or with Dist A (10 µM, Lane 3). Lane 1 represents the radiolabeled probe in the absence of incubation with protein. (* represents the HMGA1-DNA complex in Lane 2 which is diminished in intensity following addition of Dist A in Lane 3). Binding studies were repeated at least two separate times. <b>D</b>. Chromatin Immunoprecipitation (ChIP) was performed on murine bEnd.3 endothelial cells without (Lane 1) or with 3 hours of exposure to Vehicle (Veh, Upper Panel and Lanes 2–3 Lower Panel), mTNF-α (TNF, Lane 4), Distamycin A (Dist A, Lane 5), mTNF-α/Vehicle (T+V, Lane 6), and mTNF-α/Dist A (T+D, Lane 7). Immunoprecipitation of crosslinked, sonicated cell lysates was carried out with an affinity-purified HMGA1 antibody (Upper panel and Lanes 3–7 Lower Panel) or IgG rabbit control antibody (Lanes 1–2 Lower Panel), with PCR amplification using primers spanning a 246-basepair AT-rich region of the P-selectin promoter (including basepairs −542 to −521; “Promoter” in Upper Panel and all lanes in Lower Panel) or 200-basepair fragments spanning a coding region or upstream promoter region of P-selectin (“Exon” and “Upstream Promoter”, respectively in Upper Panel; see sequences in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010656#s2" target="_blank">Methods</a> section). PCR amplification was undertaken for immunoprecipitated DNA as well as for “input” DNA as an additional control for each condition. Quantitation of precipitated DNA relative to input DNA was undertaken using Quantity One 1-D Analysis Software (Bio-Rad). This experiment was performed two separate times. (*p<0.05, for increase in binding for Veh (HMGA1 Ab) compared with IgG control; **p<0.05, for reduction in binding for TNF-α/Dist compared with TNF-α/Vehicle).</p

Distamycin A disrupts binding of an inducible protein-DNA complex containing NF-κB to an AT-rich region of the P-selectin promoter.

<p><b>A.</b> As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010656#pone-0010656-g003" target="_blank">Fig. 3B</a>, lung tissue was harvested from wild type mice two hours following treatment with Vehicle (Veh), LPS/Vehicle, or LPS/Dist A (Dist), then subjected to RNA extraction. The same blot from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010656#pone-0010656-g003" target="_blank">Fig. 3B</a> was hybridized with a radiolabeled probe for HMGA1 (and an 18S probe as loading control). This experiment was repeated two separate times. <b>B.</b> Nuclear extracts from BAEC cells (Lanes 1–7) and primary murine lung endothelial cells (MLEC, Lanes 8–12) (“Nuc Ext”) with (Lanes 3–7, 9–12) or without (Lanes 2,8) TNF-α stimulation were subjected to electrophoretic mobility shift assays (EMSA) using a radiolabeled probe spanning the AT-rich region of the P-selectin promoter (basepairs −542 to −521). Lane 1 represents the radiolabeled probe without addition of nuclear extract. TNF-α-treated nuclear extract was additionally incubated and electrophoresed with the radiolabeled probe and vehicle (V, lanes 3 and 10 (or in the presence of TNF-α without vehicle, Lane 9)) or increasing concentrations of Dist A (D₁ = 10 µM, D₂ = 20 µM, Lanes 4–5, 11–12) as well as with an identical competitor (IC, Lane 6) and a non-identical competitor (NIC, Lane 7). (* represents the inducible, specific complex seen following TNF-α treatment; “→” represents disruption of the TNF-α-inducible complex following addition of Dist A (Lanes 4–5 compared with Lane 3 and Lanes 11–12 compared with Lane 9–10). All of the binding studies were repeated at least two separate times. <b>C.</b> Nuclear extracts from BAEC cells (“Nuc Ext”) with (Lanes 3–7) or without (Lane 2) TNF-α stimulation were subjected to electrophoretic mobility shift assays (EMSA) using a radiolabeled probe spanning the AT-rich region of the P-selectin promoter (basepairs −542 to −521). Lane 1 represents the radiolabeled probe without addition of nuclear extract. TNF-α-treated nuclear extract was additionally incubated and electrophoresed with the radiolabeled probe and antibodies to the NF-κB family members p50 and p65 (lanes 4–5), or unrelated and control antibodies (Ets-1 and IgG control respectively, lanes 6–7). (* represents the inducible, specific complex seen following TNF-α treatment; “←” represents supershifted band/disruption of the TNF-α-inducible complex following addition of p50 and p65 antibodies (Lanes 4-5 compared with Lanes 6–7). All of the binding studies were repeated at least two separate times.</p

Distamycin A selectively decreases induction of P-selectin promoter activity and expression.

<p><b>A.</b> BAEC cells were transiently transfected with promoter-reporter constructs for P-selectin and E-selectin. Transfected cells were treated with Vehicle, TNF-α/Vehicle, or TNF-α/Dist A, then harvested twelve hours after treatment and assessed for luciferase activity (with normalization for β-galactosidase levels). Similar experiments were performed in which transfected cells were treated with Vehicle, LPS/Vehicle, or LPS/Dist A and harvested four hours after treatment. Fold change was assessed relative to normalized values of “1” for the Vehicle-treated condition for each construct. These experiments were repeated 3 separate times with duplicate wells for each condition. (*p<0.05 compared with Vehicle for each construct; **p<0.05 compared with TNF-α/Vehicle or LPS/Vehicle for P-selectin). <b>B.</b> Lung tissue was harvested from wild type mice two hours following treatment with Vehicle (Veh), LPS/Vehicle, or LPS/Dist A (Dist), then subjected to RNA extraction and Northern blotting using a radiolabeled probe for P-selectin or E-selectin (and an 18S probe as loading control). This experiment was repeated two separate times.</p