David K. Ann

Interactions Between β-Catenin and Transforming Growth Factor-β Signaling Pathways Mediate Epithelial-Mesenchymal Transition and Are Dependent on the Transcriptional Co-activator cAMP-response Element-binding Protein (CREB)-binding Protein (CBP)

Background: Direct evidence for molecular interdependence between transforming growth factor-β (TGF-β) and Wnt pathways in mesenchymal gene regulation during epithelial-mesenchymal transition (EMT) is limited. Results: TGF-β induction of α-smooth muscle actin (α-SMA) involves ternary complex formation among Smad3, β-catenin, and CBP. Conclusion: TGF-β and β-catenin/CBP-dependent pathways coordinately regulate α-SMA induction. Significance: Inhibition of β-catenin/CBP-dependent effects of TGF-β suggests a novel therapeutic approach to EMT/fibrosis. Interactions between transforming growth factor-β (TGF-β) and Wnt are crucial to many biological processes, although specific targets, rationale for divergent outcomes (differentiation versus block of epithelial proliferation versus epithelial-mesenchymal transition (EMT)) and precise mechanisms in many cases remain unknown. We investigated β-catenin-dependent and transforming growth factor-β1 (TGF-β1) interactions in pulmonary alveolar epithelial cells (AEC) in the context of EMT and pulmonary fibrosis. We previously demonstrated that ICG-001, a small molecule specific inhibitor of the β-catenin/CBP (but not β-catenin/p300) interaction, ameliorates and reverses pulmonary fibrosis and inhibits TGF-β1-mediated α-smooth muscle actin (α-SMA) and collagen induction in AEC. We now demonstrate that TGF-β1 induces LEF/TCF TOPFLASH reporter activation and nuclear β-catenin accumulation, while LiCl augments TGF-β-induced α-SMA expression, further confirming co-operation between β-catenin- and TGF-β-dependent signaling pathways. Inhibition and knockdown of Smad3, knockdown of β-catenin and overexpression of ICAT abrogated effects of TGF-β1 on α-SMA transcription/expression, indicating a requirement for β-catenin in these Smad3-dependent effects. Following TGF-β treatment, co-immunoprecipitation demonstrated direct interaction between endogenous Smad3 and β-catenin, while chromatin immunoprecipitation (ChIP)-re-ChIP identified spatial and temporal regulation of α-SMA via complex formation among Smad3, β-catenin, and CBP. ICG-001 inhibited α-SMA expression/transcription in response to TGF-β as well as α-SMA promoter occupancy by β-catenin and CBP, demonstrating a previously unknown requisite TGF-β1/β-catenin/CBP-mediated pro-EMT signaling pathway. Clinical relevance was shown by β-catenin/Smad3 co-localization and CBP expression in AEC of IPF patients. These findings suggest a new therapeutic approach to pulmonary fibrosis by specifically uncoupling CBP/catenin-dependent signaling downstream of TGF-β.

Novel Roles for Protein Kinase Cδ-dependent Signaling Pathways in Acute Hypoxic Stress-induced Autophagy

Macroautophagy, a tightly orchestrated intracellular process for bulk degradation of cytoplasmic proteins or organelles, is believed to be essential for cell survival or death in response to stress conditions. Recent observations indicate that autophagy is an adaptive response in cells subjected to prolonged hypoxia. However, the signaling mechanisms that activate autophagy under acute hypoxic stress are not clearly understood. In this study, we show that acute hypoxic stress by treatment with 1% O2 or desferroxamine, a hypoxia-mimetic agent, of cells renders a rapid induction of LC3-II level changes and green fluorescent protein-LC3 puncta accumulation, hallmarks of autophagic processing, and that this process involves protein kinase Cδ (PKCδ), and occurs prior to the induction of BNIP3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3). Interestingly, hypoxic stress leads to a rapid and transient activation of JNK in Pa-4 or mouse embryo fibroblast cells. Acute hypoxic stress-induced changes in LC3-II level and JNK activation are attenuated in Pa-4 cells by dominant negative PKCδKD or in mouse embryo fibroblast/PKCδ-null cells. Intriguingly, the requirement of PKCδ is not apparent for starvation-induced autophagy. The importance of PKCδ in hypoxic stress-induced adaptive responses is further supported by our findings that inhibition of PKCδ-facilitated autophagy by 3-methyladenine or Atg5 knock-out renders a greater prevalence of cell death following prolonged desferroxamine treatment, whereas PKCδ- or JNK1-deficient cells exhibit resistance to extended hypoxic exposure. These results uncover dual roles of PKCδ-dependent signaling in the cell fate determination upon hypoxic exposure.

Molecular cloning of a novel protein regulated by opioid treatment of NG108-15 cells

A new cDNA clone, NGD5, has been identified from a subtraction cDNA library constructed with mRNA isolated from control neuroblastoma x glioma NG108-15 cells and cells treated for 48 h with the delta-opioid agonist, D-Ala2, D-Leu5 enkephalin (DADLE). NGD5 mRNA is decreased, in a naloxone-reversible manner, upon long-term treatment of NG108-15 cells with DADLE, indicating that this clone may be related to opioid receptor function. Northern analysis indicates that NGD5 mRNA is expressed in rat brain. Two similar nearly full-length NGD5 clones, NGD5A and NGD5B, were isolated from a lambda gt10 NG108-15 cDNA library and sequenced. The predicted 40-kDa peptide encoded for by the NGD5 cDNA has no significant homology to the recently cloned mu, delta or kappa opioid receptors nor to any other known proteins.

Isolation of a cDNA encoding a novel zinc-finger protein from neuroblastoma x glioma NG108-15 cells

A subtraction cDNA library was constructed from control hybrid NG108-15 (mouse neuroblastoma x rat glioma) cells and NG108-15 cells which had been treated for 48 h with the delta-opioid agonist D-Ala2-D-Leu5 enkephalin (DADLE) to down-regulate the delta-opioid receptor on these cells. Among the clones isolated from this library was NGD16-4, a 2768-bp clone encoding a putative 64-kDa protein containing 14 tandemly repeated zinc fingers (Zf) with high homology to the Krüppel family of Zf proteins. NGD16-4 also contains a region homologous to the A element of the Krüppel Associated Box (KRAB) domain, a domain recently linked to transcriptional repression. Southern and Northern analyses indicate that NGD16-4 is derived from the mouse genome. Northern analysis also demonstrates that expression of NGD16-4 mRNA is much higher in several mouse neuroblastoma cell lines than in mouse brain or other tissues. Although the function of NGD16-4 is unclear, the expression pattern of NGD16-4 indicates a possible association with the processes of differentiation or transformation in the mouse.

Characterization of human and rat immortalized clones of parotid acinar cells with respect to specific proteins and their mRNAs, and receptor-linked adenylate cyclase

SummaryThis study reports the isolation and characterization of a rat nontumorigenic parotid acinar cell clone (2RSG), a human nontumorigenic parotid acinar cell clone (2HPC8), and a human tumorigenic acinar clone (2HP1G). The levels ofα-amylase mRNAs detected when usingα-amylase cDNA of 1176 and 702 bp for hybridization were higher in 2RSG and 2HPC8 cells than their respective whole parotid glands. The level of these mRNAs decreased in 2HP1G cells. In contrast toα-amylase mRNAs levels, theα-amylase activity in cultured acinar cells was extremely low in comparison to whole glands, irrespective of species or cell status. The levels of proline-rich protein (PRP) mRNA and parotid secretory protein (PSP) mRNA detected when using PRP cDNA of 600 bp and PSP cDNA of 805 bp for hybridization were higher in 2RSG cells than those in rat parotid glands; the reverse was observed in 2HPC8 cells and human parotid glands. The levels of PRP mRNA and PSP mRNA in 2HPC8 and 2HP1G acinar cells were similar. The level of mRNA was not detectable in murine neuroblastoma cells (NBP2) using the sameα-amylase cDNA, PRP cDNA and PSP cDNA for hybridization. The PSP level in rat parotid gland was lower than that found in 2RSG cells; the reverse was observed in 2HPC8 cells and human parotid glands. The level of PSP in 2HP1G cells was higher than that found in 2HPC8 cells. Isoproterenol increased the cAMP level in 2RSG, 2HPC8, and 2HP1G clones, being most effective in 2RSG cells, and least effective in 2HPG cells. Prostaglandin E1 (PGE1) also increased cAMP level, being most effective in 2HPC8 cells and ineffective in 2HP1G cells, suggesting that the PGE1 receptor-linked adenylate cyclase becomes inactive upon transformation. These results suggest that the three clonal acinar cells from rat and human parotid glands reported here can be useful in comparative studies on regulation of growth, differentiation, and transformation.

Macaque Salivary Proline-Rich Protein: Structure, Evolution, and Expression

Proline-rich proteins are a family of proteins that exhibit unique features including an unusual high proline content and salivary-specificity. As a major constituent in the salivary secretion of higher primates, proline-rich proteins may have biological roles in oral lubrication and protection. In this article, the genomic structure and regulation by cAMP of one of the macaque salivary proline-rich protein genes, MnP4, is reviewed. The evolution of this multigene family of proteins is also discussed.