Faculty Bibliography

We report recessive mutations in the gene for the latent transforming growth factor-beta binding protein 4 (LTBP4) in four unrelated patients with a human syndrome disrupting pulmonary, gastrointestinal, urinary, musculoskeletal, craniofacial, and dermal development. All patients had severe respiratory distress, with cystic and atelectatic changes in the lungs complicated by tracheomalacia and diaphragmatic hernia. Three of the four patients died of respiratory failure. Cardiovascular lesions were mild, limited to pulmonary artery stenosis and patent foramen ovale. Gastrointestinal malformations included diverticulosis, enlargement, tortuosity, and stenosis at various levels of the intestinal tract. The urinary tract was affected by diverticulosis and hydronephrosis. Joint laxity and low muscle tone contributed to musculoskeletal problems compounded by postnatal growth delay. Craniofacial features included microretrognathia, flat midface, receding forehead, and wide fontanelles. All patients had cutis laxa. Four of the five identified LTBP4 mutations led to premature termination of translation and destabilization of the LTBP4 mRNA. Impaired synthesis and lack of deposition of LTBP4 into the extracellular matrix (ECM) caused increased transforming growth factor-beta (TGF-beta) activity in cultured fibroblasts and defective elastic fiber assembly in all tissues affected by the disease. These molecular defects were associated with blocked alveolarization and airway collapse in the lung. Our results show that coupling of TGF-beta signaling and ECM assembly is essential for proper development and is achieved in multiple human organ systems by multifunctional proteins such as LTBP4

BACKGROUND: Phosphorylation is a key regulatory event in controlling the function of the cardiac gap junction protein connexin43 (Cx43). Three new phosphorylation sites (S296, S297, S306) have been identified on Cx43; two of these sites (S297 and S306) are dephosphorylated during ischemia. The functional significance of these new sites is currently unknown. OBJECTIVE: The purpose of this study was to examine the role of S296, S297, and S306 in the regulation of electrical intercellular communication. METHODS: To mimic constitutive dephosphorylation, serine was mutated to alanine at the three sites and expressed in HeLa cells. Electrical coupling and single channel measurements were performed by double patch clamp. Protein expression levels were assayed by western blotting, localization of Cx43, and phosphorylation of S306 by immunolabeling. Free hemichannels were assessed by biotinylation. RESULTS: Macroscopic conductance in cells expressing S306A was reduced to 57% compared to wild type (WT), whereas coupling was not significantly changed in cells expressing either S296A or S297A. S306A-expressing cells displayed similar protein and free hemichannel abundance compared to WT Cx43, whereas the fractional area of plaques in cell-to-cell interfaces was increased. However, single channel measurements showed a WT Cx43 main state conductance of 119 pS, whereas the main state conductance of S306A channels was reduced to 95 pS. Furthermore, channel gating was affected in S306A channels. CONCLUSION: Lack of phosphorylation at serine 306 results in reduced coupling, which can be explained by reduced single channel conductance. We suggest that dephosphorylation of S306 partly explains the electrical uncoupling seen in myocardial ischemia

The establishment of sexual identity is a crucial step of germ cell development in sexually reproducing organisms. Sex determination in the germline is controlled differently than in the soma, and often depends on communication from the soma. To investigate how sexual identity is established in the Drosophila germline, we first conducted a molecular screen for genes expressed in a sex-specific manner in embryonic germ cells. Sex-specific expression of these genes is initiated at the time of gonad formation (stage 15), indicating that sexual identity in the germline is established by this time. Experiments where the sex of the soma was altered relative to that of the germline (by manipulating transformer) reveal a dominant role for the soma in regulating initial germline sexual identity. Germ cells largely take on the sex of the surrounding soma, although the sex chromosome constitution of the germ cells still plays some role at this time. The male soma signals to the germline through the JAK/STAT pathway, while the nature of the signal from the female soma remains unknown. We also find that the genes ovo and ovarian tumor (otu) are expressed in a female-specific manner in embryonic germ cells, consistent with their role in promoting female germline identity. However, removing the function of ovo and otu, or reducing germline function of Sex lethal, had little effect on establishment of germline sexual identity. This is consistent with our findings that signals from the soma are dominant over germline autonomous cues at the initial stage of germline sex determination.

While both the adrenal medulla and sympathetic nervous system are important in mediating the catecholaminergic response to stress, there are crucial differences in the mechanism. Stress elevates tyrosine hydroxylase (TH) protein and mRNA levels in both the adrenal medulla and sympathetic ganglia. In the adrenal medulla, transcription of the TH gene is rapidly induced with immobilization (IMO) stress. Here, we examine whether IMO also increases TH transcription in the superior cervical ganglia (SCG). Quantitative real-time reverse transcription polymerase chain reaction was used to determine the changes in TH mRNA and in transcripts containing intron 2. As expected in the adrenal medulla following repeated IMO TH mRNA and intron containing transcripts were elevated about 5-fold. In the SCG, a significant increase in TH mRNA was observed following repeated 2 h IMO for 2 or 6 days, but not with single IMO. The intron 2 containing transcripts were elevated about 50% above controls with even single IMO, and were at similarly elevated levels after the 2nd or 6th repeated daily IMO. The results indicate, for the first time, that transcriptional mechanisms are involved in mediating the IMO stress triggered elevation in TH gene expression in the SCG.

Molecular events involved in successful embryo implantation are not well understood. In this study, we used MALDI imaging mass spectrometry (IMS) technologies to characterize the spatial and temporal distribution of phospholipid species associated with mouse embryo implantation. Molecular images showing phospholipid distribution within implantation sites changed markedly between distinct cellular areas during days 4-8 of pregnancy. For example, by day 8, linoleate- and docosahexaenoate-containing phospholipids localized to regions destined to undergo cell death, whereas oleate-containing phospholipids localized to angiogenic regions. Arachidonate-containing phospholipids showed different segregation patterns depending on the lipid class, revealing a strong correlation of phosphatidylethanolamines and phosphatidylinositols with cytosolic phospholipase A(2alpha) and cyclooxygenase-2 during embryo implantation. LC-ESI-MS/MS was used to validate MALDI IMS phospholipid distribution patterns. Overall, molecular images revealed the dynamic complexity of lipid distributions in early pregnancy, signifying the importance of complex interplay of lipid molecules in uterine biology and implantation.

OBJECTIVE: Transgenic expression of diacylglycerol acyltransferase-1 (DGAT1) in skeletal muscle leads to protection against fat-induced insulin resistance despite accumulation of intramuscular triglyceride, a phenomenon similar to what is known as the "athlete paradox." The primary objective of this study is to determine how DGAT1 affects muscle fatty acid oxidation in relation to whole-body energy metabolism and insulin sensitivity. RESEARCH DESIGN AND METHODS: We first quantified insulin sensitivity and the relative tissue contributions to the improved whole-body insulin sensitivity in muscle creatine kisase (MCK)-DGAT1 transgenic mice by hyperinsulinemic-euglycemic clamps. Metabolic consequences of DGAT1 overexpression in skeletal muscles were determined by quantifying triglyceride synthesis/storage (anabolic) and fatty acid oxidation (catabolic), in conjunction with gene expression levels of representative marker genes in fatty acid metabolism. Whole-body energy metabolism including food consumption, body weights, oxygen consumption, locomotor activity, and respiration exchange ratios were determined at steady states. RESULTS: MCK-DGAT1 mice were protected against muscle lipoptoxicity, although they remain susceptible to hepatic lipotoxicity. While augmenting triglyceride synthesis, DGAT1 overexpression also led to increased muscle mitochondrial fatty acid oxidation efficiency, as compared with wild-type muscles. On a high-fat diet, MCK-DGAT1 mice displayed higher basal metabolic rates and 5-10% lower body weights compared with wild-type littermates, whereas food consumption was not different. CONCLUSIONS: DGAT1 overexpression in skeletal muscle led to parallel increases in triglyceride synthesis and fatty acid oxidation. Seemingly paradoxical, this phenomenon is characteristic of insulin-sensitive myofibers and suggests that DGAT1 plays an active role in metabolic "remodeling" of skeletal muscle coupled with insulin sensitization.

Benzo[a]pyrene (B[a]P) is a ligand for the aryl hydrocarbon receptor (Ahr). After binding ligand, Ahr dimerizes with the aryl hydrocarbon receptor nuclear translocator (Arnt) protein, and the dimer upregulates the transcription of Cyp1a1, Cyp1b1 and other enzymes involved in the metabolic activation of B[a]P. Arnt null mice die in utero. Mice in which Arnt deletion occurs constitutively in the epidermis die perinatally. In the current study, mice were developed in which the Arnt gene could be deleted specifically in adult skin epidermis. This deletion had no overt pathological effect. Homozygosity for a null reduced nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase allele was introduced into the above mouse strain to render it more susceptible to tumor initiation by B[a]P. Deletion of Arnt in the epidermis of this strain completely prevented the induction of skin tumors in a tumor initiation-promotion protocol in which a single topical application of B[a]P acted as the tumor-initiating event, and tumor promotion was provided by repeated topical applications of 12-O-tetradecanoyl phorbol-13-acetate (TPA). In contrast, deletion of Arnt did not prevent the induction of skin tumors in a protocol also using TPA as the promoter but using as the initiator N-methyl-N'-nitro-N-nitrosoguanidine, whose activity is unlikely to be affected by the activity of Ahr, Arnt or their target genes. These observations demonstrate that Arnt is required for tumor initiation by B[a]P in this system.

Parathyroid hormone-related protein (PTHrP) acts on the mammary mesenchyme and is required for proper embryonic mammary development. In order to understand PTHrP's effects on mesenchymal cells, we profiled gene expression in WT and PTHrP(-/-) mammary buds, and in WT and K14-PTHrP ventral skin at E15.5. By cross-referencing the differences in gene expression between these groups, we identified 35 genes potentially regulated by PTHrP in the mammary mesenchyme, including 6 genes known to be involved in BMP signaling. One of these genes was MMP2. We demonstrated that PTHrP and BMP4 regulate MMP2 gene expression and MMP2 activity in mesenchymal cells. Using mammary bud cultures, we demonstrated that MMP2 acts downstream of PTHrP to stimulate ductal outgrowth. Future studies on the functional role of other genes on this list should expand our knowledge of how PTHrP signaling triggers the onset of ductal outgrowth from the embryonic mammary buds.

The isthmic organizer and its key effector molecule, fibroblast growth factor 8 (Fgf8), have been cornerstones in studies of how organizing centers differentially pattern tissues. Studies have implicated different levels of Fgf8 signaling from the mid/hindbrain boundary (isthmus) as being responsible for induction of different structures within the tectal-isthmo-cerebellum region. However, the role of Fgf8 signaling for different durations in patterning tissues has not been studied. To address this, we conditionally ablated Fgf8 in the isthmus and uncovered that prolonged expression of Fgf8 is required for the structures found progressively closer to the isthmus to form. We found that cell death cannot be the main factor accounting for the loss of brain structures near the isthmus, and instead demonstrate that tissue transformation underlies the observed phenotypes. We suggest that the remaining Fgf8 and Fgf17 signaling in our temporal Fgf8 conditional mutants is sufficient to ensure survival of most midbrain/hindbrain cells near the isthmus. One crucial role for sustained Fgf8 function is in repressing Otx2 in the hindbrain, thereby allowing the isthmus and cerebellum to form. A second requirement for sustained Fgf8 signaling is to induce formation of a posterior tectum. Finally, Fgf8 is also required to maintain the borders of expression of a number of key genes involved in tectal-isthmo-cerebellum development. Thus, the duration as well as the strength of Fgf8 signaling is key to patterning of the mid/hindbrain region. By extrapolation, the length of Fgf8 expression could be crucial to Fgf8 function in other embryonic organizers

In Alzheimer's disease, microglia cluster around beta-amyloid deposits, suggesting that these cells are important for amyloid plaque formation, maintenance and/or clearance. We crossed two distinct APP transgenic mouse strains with CD11b-HSVTK mice, in which nearly complete ablation of microglia was achieved for up to 4 weeks after ganciclovir application. Neither amyloid plaque formation and maintenance nor amyloid-associated neuritic dystrophy depended on the presence of microglia