Differential growth regulation by beta-adrenergic/cyclic amp signaling in phenotypically different human pulmonary adenocarcinomas cell lines that express the epidermal growth factor-mediated pathway

Human pulmonary adenocarcinoma (PAC), which originates from bronchiolar epitheliaVClara cells (P ACC) or the alveolar type II cells (PAC type II), constitutes one of the most rapidly rising lung cancer types. The occurrence of PAC in smoking and nonsmoking individuals suggests that other factors besides smoking contribute to the development and progression of PAC. Earlier studies showed the presence of beta-adrenergic/cAMP growth pathway in PACC. Consequently, beta-adrenergic stimulants in various drug formulations that are used in the management of chronic respiratory diseases have been proposed as potential risk factors for the development of PAC. On the other hand, little is known about the role of this pathway in the proliferation of PAC type II. Using thymidine incorporation, this study showed that the two PAC phenotypes have differential response to the betaadrenergic/cAMP stimulation. Isoproterenol (broad-spectrum beta-adrenergic agonist) and forskolin (adenylyl cyclase stimulator) induced significant proliferative response in NCI-H322 cell line (PACC). Both beta-1 and beta-2 adrenergic receptors and cAMP are involved in this proliferative response as shown by selective receptor and enzyme inhibitors. On the other hand, A549 cells (PAC-type II) were inhibited by forskolin while being unresponsive to beta-adrenergic stimulation. Accordingly, forskolin caused significant and persistent activation of the extracellular signal regulated kinase (ERK.1/2) in NCI-H322 cells in contrast to A549 cells, in which inhibition was observed. Cyclic AMP immunoassay of basal and stimulant-induced cAMP amount showed marked difference between the two cell lines. The basal cAMP content in A549 cells was significantly lower than that of NCI-H322 cells. Moreover, isoproterenol had no effect while forskolin had significant but moderate rise of cAMP. By contrast, both isoproterenol and forskolin induced a marked and significant accumulation of cAMP in NCI-H322 cells. In both cell lines, significantly higher proliferative response was observed in low serum than in high serum culture condition. The epidermal growth factor receptor (EGFR)­ mediated growth pathway is common to both cell lines as demonstrated by the use of AG 1478 (EGFR-specific tyrosine kinase inhibitor). Furthermore, AG 1478 inhibited isoproterenol but not forskolin-induced proliferation in NCI-H322 cells, implying the existence of cAMP dependent and independent growth pathways and transactivation of the EGFR. The present study demonstrated an important difference between two cancer cell phenotypes that are generally grouped as PAC. The implication of this distinction in relation to cancer chemoprevention approaches and chronic management of respiratory diseases with beta-adrenergic stimulants was emphasized. On the other hand, the EGFR growth pathway is commonly expressed and plays a central role in the proliferation of both PAC phenotypes. Consequently, the EGFR pathway may provide a common chemotherapeutic target in broad family of cancer types and phenotypes with diverse growth pathways

Factors necessary to produce basoapical polarity in human glandular epithelium formed in conventional and high-throughput three-dimensional culture: example of the breast epithelium

<p>Abstract</p> <p>Background</p> <p>Basoapical polarity in epithelia is critical for proper tissue function, and control of proliferation and survival. Cell culture models that recapitulate epithelial tissue architecture are invaluable to unravel developmental and disease mechanisms. Although factors important for the establishment of basal polarity have been identified, requirements for the formation of apical polarity in three-dimensional tissue structures have not been thoroughly investigated.</p> <p>Results</p> <p>We demonstrate that the human mammary epithelial cell line-3522 S1, provides a resilient model for studying the formation of basoapical polarity in glandular structures. Testing three-dimensional culture systems that differ in composition and origin of substrata reveals that apical polarity is more sensitive to culture conditions than basal polarity. Using a new high-throughput culture method that produces basoapical polarity in glandular structures without a gel coat, we show that basal polarity-mediated signaling and collagen IV are both necessary for the development of apical polarity.</p> <p>Conclusion</p> <p>These results provide new insights into the role of the basement membrane, and especially collagen IV, in the development of the apical pole, a critical element of the architecture of glandular epithelia. Also, the high-throughput culture method developed in this study should open new avenues for high-content screening of agents that act on mammary tissue homeostasis and thus, on architectural changes involved in cancer development.</p

MacroH2A1 isoforms are associated with epigenetic markers for activation of lipogenic genes in fat-induced steatosis.

The importance of epigenetic changes in the development of hepatic steatosis is largely unknown. The histone variant macroH2A1 under alternative splicing gives rise to macroH2A1.1 and macroH2A1.2. In this study, we show that the macroH2A1 isoforms play an important role in the regulation of lipid accumulation in hepatocytes. Hepatoma cell line and immortalized human hepatocytes transiently transfected or knocked down with macroH2A1 isoforms were used as in vitro model of fat-induced steatosis. Gene expressions were analyzed by quantitative PCR array and Western blot. Chromatin immunoprecipitation analysis was performed to check the association of histone H3 lysine 27 trimethylation (H3K27me3) and histone H3 lysine 4 trimethylation (H3K4me3) with the promoter of lipogenic genes. Livers from knockout mice that are resistant to lipid deposition despite a high-fat diet were used for histopathology. We found that macroH2A1.2 is regulated by fat uptake and that its overexpression caused an increase in lipid uptake, triglycerides, and lipogenic genes compared with macroH2A1.1. This suggests that macroH2A1.2 is important for lipid uptake, whereas macroH2A1.1 was found to be protective. The result was supported by a high positivity for macroH2A1.1 in knockout mice for genes targeted by macroH2A1 (Atp5a1 and Fam73b), that under a high-fat diet presented minimal lipidosis. Moreover, macroH2A1 isoforms differentially regulate the expression of lipogenic genes by modulating the association of the active (H3K4me3) and repressive (H3K27me3) histone marks on their promoters. This study underlines the importance of the replacement of noncanonical histones in the regulation of genes involved in lipid metabolism in the progression of steatosis

Genome-wide screening of mouse knockouts reveals novel genes required for normal integumentary and oculocutaneous structure and function.

Oculocutaneous syndromes are often due to mutations in single genes. In some cases, mouse models for these diseases exist in spontaneously occurring mutations, or in mice resulting from forward mutatagenesis screens. Here we present novel genes that may be causative for oculocutaneous disease in humans, discovered as part of a genome-wide screen of knockout-mice in a targeted single-gene deletion project. The International Mouse Phenotyping Consortium (IMPC) database (data release 10.0) was interrogated for all mouse strains with integument abnormalities, which were then cross-referenced individually to identify knockouts with concomitant ocular abnormalities attributed to the same targeted gene deletion. The search yielded 307 knockout strains from unique genes with integument abnormalities, 226 of which have not been previously associated with oculocutaneous conditions. Of the 307 knockout strains with integument abnormalities, 52 were determined to have ocular changes attributed to the targeted deletion, 35 of which represent novel oculocutaneous genes. Some examples of various integument abnormalities are shown, as well as two examples of knockout strains with oculocutaneous phenotypes. Each of the novel genes provided here are potentially relevant to the pathophysiology of human integumentary, or oculocutaneous conditions, such as albinism, phakomatoses, or other multi-system syndromes. The novel genes reported here may implicate molecular pathways relevant to these human diseases and may contribute to the discovery of novel therapeutic targets

Tankyrase represses autoinflammation through the attenuation of TLR2 signaling

Dysregulation of Toll-like receptor (TLR) signaling contributes to the pathogenesis of autoimmune diseases. Here, we provide genetic evidence that tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, negatively regulates TLR2 signaling. We show that mice lacking tankyrase in myeloid cells developed severe systemic inflammation with high serum inflammatory cytokine levels. We provide mechanistic evidence that tankyrase deficiency resulted in tyrosine phosphorylation and activation of TLR2 and show that phosphorylation of tyrosine 647 within the TIR domain by SRC and SYK kinases was critical for TLR2 stabilization and signaling. Last, we show that the elevated cytokine production and inflammation observed in mice lacking tankyrase in myeloid cells were dependent on the adaptor protein 3BP2, which is required for SRC and SYK activation. These data demonstrate that tankyrase provides a checkpoint on the TLR-mediated innate immune response

Erratum: Author Correction: Identification of genes required for eye development by high-throughput screening of mouse knockouts.

[This corrects the article DOI: 10.1038/s42003-018-0226-0.]

Identification of genes required for eye development by high-throughput screening of mouse knockouts.

Despite advances in next generation sequencing technologies, determining the genetic basis of ocular disease remains a major challenge due to the limited access and prohibitive cost of human forward genetics. Thus, less than 4,000 genes currently have available phenotype information for any organ system. Here we report the ophthalmic findings from the International Mouse Phenotyping Consortium, a large-scale functional genetic screen with the goal of generating and phenotyping a null mutant for every mouse gene. Of 4364 genes evaluated, 347 were identified to influence ocular phenotypes, 75% of which are entirely novel in ocular pathology. This discovery greatly increases the current number of genes known to contribute to ophthalmic disease, and it is likely that many of the genes will subsequently prove to be important in human ocular development and disease

Identification of genes required for eye development by high-throughput screening of mouse knockouts

International audienc

Paracrine and gap junctional communication in breast epithelial cells: Role in diffrentiation and cancer

Homeostasis of the mammary gland is closely regulated by systemic hormones and local factors. It is documented that the effect of systemic hormones is mediated and modulated by local diffusible factors that are communicated between the various cells of the mammary gland. Consequently, local cellular interactions, notably paracrine and gap junctional communication (GJC), play critical roles in normal function as well as dysfunction associated with cancer within the tissue context of the mammary gland. This thesis addressed the potential role of these cellular interactions in differentiation and tumor progression of human mammary epithelium using a three-dimensional (3D) cell culture. Early development of breast cancer is characterized by the presence of few neoplastic epithelial cells amidst an excess of normal epithelial structures. However, the influence of the normal epithelium on tumor progression is largely unknown. Towards this goal, I have employed a 3D paracrine interaction model of genetically matched phenotypically normal and malignant human mammary epithelial cells. The data show that differentiated glandular structures (acini) promoted tumor growth. Further, this effect is attributed to a mechanism that abrogates IL-6-mediated autocrine inhibitory pathway in malignant cells. This protection by the normal epithelium could favor the growth early stage of breast tumors. Gap junctional communication by connexins (Cxs) is considered essential for the differentiation of mammary epithelial cells mainly because of its tumor suppressor effect on breast cancer cells. However, the role of GJC in the differentiation of the normal epithelium, notably in the establishment of basoapical polarity, is largely unknown. To this end, I used a 3D cell culture system that promotes glandular (acinar) differentiation of human mammary luminal epithelial cells. Acini expressed apical Cx43, remarkably reproducing the distribution observed in vivo in breast tissue. Chemically blocking GJC or silencing Cx43 expression during differentiation resulted in marked acinar malformation. Glandular malformation was also accompanied by disruption of apical polarity as shown by the loss of the tight junction protein ZO-1 from the apical location. Interestingly, structures that lacked apical polarity were selectively induced to enter the cell cycle by mitogenic stimulation or by altering nuclear organization. These data show a novel role for Cx43 in the regulation of apical polarity, a critical feature of epithelial homeostasis. Overall, this work demonstrated the importance of cellular communication in differentiation and tumor progression of the mammary gland