In JoVE (1)

Other Publications (6)

Articles by Sharon J. Sequeira in JoVE

Other articles by Sharon J. Sequeira on PubMed

Inhibition of Proliferation by PERK Regulates Mammary Acinar Morphogenesis and Tumor Formation

PloS One. 2007  |  Pubmed ID: 17637831

Endoplasmic reticulum (ER) stress signaling can be mediated by the ER kinase PERK, which phosphorylates its substrate eIF2alpha. This in turn, results in translational repression and the activation of downstream programs that can limit cell growth through cell cycle arrest and/or apoptosis. These responses can also be initiated by perturbations in cell adhesion. Thus, we hypothesized that adhesion-dependent regulation of PERK signaling might determine cell fate. We tested this hypothesis in a model of mammary acini development, a morphogenetic process regulated in part by adhesion signaling. Here we report a novel role for PERK in limiting MCF10A mammary epithelial cell proliferation during acinar morphogenesis in 3D Matrigel culture as well as in preventing mammary tumor formation in vivo. We show that loss of adhesion to a suitable substratum induces PERK-dependent phosphorylation of eIF2alpha and selective upregulation of ATF4 and GADD153. Further, inhibition of endogenous PERK signaling during acinar morphogenesis, using two dominant-negative PERK mutants (PERK-DeltaC or PERK-K618A), does not affect apoptosis but results instead in hyper-proliferative and enlarged lumen-filled acini, devoid of proper architecture. This phenotype correlated with an adhesion-dependent increase in translation initiation, Ki67 staining and upregulation of Laminin-5, ErbB1 and ErbB2 expression. More importantly, the MCF10A cells expressing PERKDeltaC, but not a vector control, were tumorigenic in vivo upon orthotopic implantation in denuded mouse mammary fat pads. Our results reveal that the PERK pathway is responsive to adhesion-regulated signals and that it is essential for proper acinar morphogenesis and in preventing mammary tumor formation. The possibility that deficiencies in PERK signaling could lead to hyperproliferation of the mammary epithelium and increase the likelihood of tumor formation, is of significance to the understanding of breast cancer.

Inhibition of EIF2alpha Dephosphorylation Inhibits ErbB2-induced Deregulation of Mammary Acinar Morphogenesis

BMC Cell Biology. 2009  |  Pubmed ID: 19754954

The ErbB2/Her2/Neu receptor tyrosine kinase is amplified in approximately 30% of human breast cancers. Phosphorylation of the translation initiation factor, eIF2alpha inhibits global protein synthesis and activates a stress signaling and growth suppressive program. We have shown that forced phosphorylation of eIF2alpha can suppress head and neck, colorectal carcinoma and multiple myeloma tumor growth and/or survival. Here we explore whether ErbB2 modulates eIF2alpha phosphorylation and whether forced phosphorylation of the latter can antagonize ErbB2 deregulation of mammary acinar morphogenesis.

Identification of a Mechanochemical Checkpoint and Negative Feedback Loop Regulating Branching Morphogenesis

Developmental Biology. Dec, 2009  |  Pubmed ID: 19804774

Cleft formation is the initial step in submandibular salivary gland (SMG) branching morphogenesis, and may result from localized actomyosin-mediated cellular contraction. Since ROCK regulates cytoskeletal contraction, we investigated the effects of ROCK inhibition on mouse SMG ex vivo organ cultures. Pharmacological inhibitors of ROCK, isoform-specific ROCK I but not ROCK II siRNAs, as well as inhibitors of myosin II activity stalled clefts at initiation. This finding implies the existence of a mechanochemical checkpoint regulating the transition of initiated clefts into progression-competent clefts. Downstream of the checkpoint, clefts are rendered competent through localized assembly of fibronectin promoted by ROCK I/myosin II. Cleft progression is primarily mediated by ROCK I/myosin II-stimulated cell proliferation with a contribution from cellular contraction. Furthermore, we demonstrate that FN assembly itself promotes epithelial proliferation and cleft progression in a ROCK-dependent manner. ROCK also stimulates a proliferation-independent negative feedback loop to prevent further cleft initiations. These results reveal that cleft initiation and progression are two physically and biochemically distinct processes.

Extracellular Matrix and Growth Factors in Salivary Gland Development

Frontiers of Oral Biology. 2010  |  Pubmed ID: 20428011

The interstitial extracellular matrix (ECM) and epithelial-cell associated basement membrane (BM) play critical roles in the morphogenesis and differentiation of developing salivary glands. Early studies used ex vivo organ culture and tissue recombination methods to identify the importance of the ECM in organ development. Incorporation of transgenic mice and molecular tools has facilitated progress in our understanding of the mechanisms by which ECM proteins influence SMG development. Recent work has identified alterations in the ECM, BM, and associated proteins in salivary gland diseases, including Sjögren's syndrome and salivary gland cancers, but the significance of such changes is not known. Understanding the basic mechanisms controlling morphogenesis and differentiation in mammalian organ development is the first step towards understanding pathogenesis. Molecular characterization of the function of the ECM and BM in cellular processes is critical for future development of therapeutic approaches in regenerative medicine and tissue engineering. Here we provide a historical overview of experiments defining the functions of the ECM, ECM receptors, and associated molecules in salivary gland development. We include a discussion of the function of ECM-associated proteases and major growth factor signaling components that are in some way regulated by the ECM or associated molecules. We conclude with a discussion of defects in ECM and BM occurring in salivary gland pathologies and speculation on future areas of research pertaining to further understanding of the function of the ECM in the salivary gland.

The Regulation of Focal Adhesion Complex Formation and Salivary Gland Epithelial Cell Organization by Nanofibrous PLGA Scaffolds

Biomaterials. Apr, 2012  |  Pubmed ID: 22285464

Nanofiber scaffolds have been useful for engineering tissues derived from mesenchymal cells, but few studies have investigated their applicability for epithelial cell-derived tissues. In this study, we generated nanofiber (250 nm) or microfiber (1200 nm) scaffolds via electrospinning from the polymer, poly-l-lactic-co-glycolic acid (PLGA). Cell-scaffold contacts were visualized using fluorescent immunocytochemistry and laser scanning confocal microscopy. Focal adhesion (FA) proteins, such as phosphorylated FAK (Tyr397), paxillin (Tyr118), talin and vinculin were localized to FA complexes in adult cells grown on planar surfaces but were reduced and diffusely localized in cells grown on nanofiber surfaces, similar to the pattern observed in adult mouse salivary gland tissues. Significant differences in epithelial cell morphology and cell clustering were also observed and quantified, using image segmentation and computational cell-graph analyses. No statistically significant differences in scaffold stiffness between planar PLGA film controls compared to nanofibers scaffolds were detected using nanoindentation with atomic force microscopy, indicating that scaffold topography rather than mechanical properties accounts for changes in cell attachments and cell structure. Finally, PLGA nanofiber scaffolds could support the spontaneous self-organization and branching of dissociated embryonic salivary gland cells. Nanofiber scaffolds may therefore have applicability in the future for engineering an artificial salivary gland.

Selective Functionalization of Nanofiber Scaffolds to Regulate Salivary Gland Epithelial Cell Proliferation and Polarity

Biomaterials. Nov, 2012  |  Pubmed ID: 22938763

Epithelial cell types typically lose apicobasal polarity when cultured on 2D substrates, but apicobasal polarity is required for directional secretion by secretory cells, such as salivary gland acinar cells. We cultured salivary gland epithelial cells on poly(lactic-co-glycolic acid) (PLGA) nanofiber scaffolds that mimic the basement membrane, a specialized extracellular matrix, and examined cell proliferation and apicobasal polarization. Although cells proliferated on nanofibers, chitosan-coated nanofiber scaffolds stimulated proliferation of salivary gland epithelial cells. Although apicobasal cell polarity was promoted by the nanofiber scaffolds relative to flat surfaces, as determined by the apical localization of ZO-1, it was antagonized by the presence of chitosan. Neither salivary gland acinar nor ductal cells fully polarized on the nanofiber scaffolds, as determined by the homogenous membrane distribution of the mature tight junction marker, occludin. However, nanofiber scaffolds chemically functionalized with the basement membrane protein, laminin-111, promoted more mature tight junctions, as determined by apical localization of occludin, but did not affect cell proliferation. To emulate the multifunctional capabilities of the basement membrane, bifunctional PLGA nanofibers were generated. Both acinar and ductal cell lines responded to signals provided by bifunctional scaffolds coupled to chitosan and laminin-111, demonstrating the applicability of such scaffolds for epithelial cell types.

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