Structural investigation of the Bacillus subtilis morphogenic factor RodZ

A thesis to obtain a Master degree in Structural and Functional Biochemistry

The AP-1 transcription factor c-Jun prevents stress-imposed maladaptive remodeling of the heart.

Systemic hypertension increases cardiac workload and subsequently induces signaling networks in heart that underlie myocyte growth (hypertrophic response) through expansion of sarcomeres with the aim to increase contractility. However, conditions of increased workload can induce both adaptive and maladaptive growth of heart muscle. Previous studies implicate two members of the AP-1 transcription factor family, junD and fra-1, in regulation of heart growth during hypertrophic response. In this study, we investigate the function of the AP-1 transcription factors, c-jun and c-fos, in heart growth. Using pressure overload-induced cardiac hypertrophy in mice and targeted deletion of Jun or Fos in cardiomyocytes, we show that c-jun is required for adaptive cardiac hypertrophy, while c-fos is dispensable in this context. c-jun promotes expression of sarcomere proteins and suppresses expression of extracellular matrix proteins. Capacity of cardiac muscle to contract depends on organization of principal thick and thin filaments, myosin and actin, within the sarcomere. In line with decreased expression of sarcomere-associated proteins, Jun-deficient cardiomyocytes present disarrangement of filaments in sarcomeres and actin cytoskeleton disorganization. Moreover, Jun-deficient hearts subjected to pressure overload display pronounced fibrosis and increased myocyte apoptosis finally resulting in dilated cardiomyopathy. In conclusion, c-jun but not c-fos is required to induce a transcriptional program aimed at adapting heart growth upon increased workload.

Actin filament dynamics impacts keratinocyte stem cell maintenance.

Cultured human epidermal keratinocyte stem cells (holoclones) are crucial for regenerative medicine for burns and genetic disorders. In serial culture, holoclones progressively lose their proliferative capacity to become transient amplifying cells with limited growth (paraclones), a phenomenon termed clonal conversion. Although it negatively impacts the culture lifespan and the success of cell transplantation, little is known on the molecular mechanism underlying clonal conversion. Here, we show that holoclones and paraclones differ in their actin filament organization, with actin bundles distributed radially in holoclones and circumferentially in paraclones. Moreover, actin organization sets the stage for a differing response to epidermal growth factor (EGF), since EGF signalling induces a rapid expansion of colony size in holoclones and a significant reduction in paraclones. Furthermore, inhibition of PI3K or Rac1 in holoclones results in the reorganization of actin filaments in a pattern that is similar to that of paraclones. Importantly, continuous Rac1 inhibition in holoclones results in clonal conversion and reduction of growth potential. Together, our data connect loss of stem cells to EGF-induced colony dynamics governed by Rac1.

Zoledronate sensitizes endothelial cells to tumor necrosis factor-induced programmed cell death: evidence for the suppression of sustained activation of focal adhesion kinase and protein kinase B/Akt.

Bisphosphonates are potent inhibitors of osteoclast function widely used to treat conditions of excessive bone resorption, including tumor bone metastases. Recent evidence indicates that bisphosphonates have direct cytotoxic activity on tumor cells and suppress angiogenesis, but the associated molecular events have not been fully characterized. In this study we investigated the effects of zoledronate, a nitrogen-containing bisphosphonate, and clodronate, a non-nitrogen-containing bisphosphonate, on human umbilical vein endothelial cell (HUVEC) adhesion, migration, and survival, three events essential for angiogenesis. Zoledronate inhibited HUVEC adhesion mediated by integrin alphaVbeta3, but not alpha5beta1, blocked migration and disrupted established focal adhesions and actin stress fibers without modifying cell surface integrin expression level or affinity. Zoledronate treatment slightly decreased HUVEC viability and strongly enhanced tumor necrosis factor (TNF)-induced cell death. HUVEC treated with zoledronate and TNF died without evidence of enhanced annexin-V binding, chromatin condensation, or nuclear fragmentation and caspase dependence. Zoledronate inhibited sustained phosphorylation of focal adhesion kinase (FAK) and in combination with TNF, with and without interferon (IFN) gamma, of protein kinase B (PKB/Akt). Constitutive active PKB/Akt protected HUVEC from death induced by zoledronate and TNF/IFNgamma. Phosphorylation of c-Src and activation of NF-kappaB were not affected by zoledronate. Clodronate had no effect on HUVEC adhesion, migration, and survival nor did it enhanced TNF cytotoxicity. Taken together these data demonstrate that zoledronate sensitizes endothelial cells to TNF-induced, caspase-independent programmed cell death and point to the FAK-PKB/Akt pathway as a novel zoledronate target. These results have potential implications to the clinical use of zoledronate as an anti-angiogenic or anti-cancer agent.

Loss of serum response factor in keratinocytes results in hyperproliferative skin disease in mice.

The transcription factor serum response factor (SRF) plays a crucial role in the development of several organs. However, its role in the skin has not been explored. Here, we show that keratinocytes in normal human and mouse skin expressed high levels of SRF but that SRF expression was strongly downregulated in the hyperproliferative epidermis of wounded and psoriatic skin. Keratinocyte-specific deletion within the mouse SRF locus during embryonic development caused edema and skin blistering, and all animals died in utero. Postnatal loss of mouse SRF in keratinocytes resulted in the development of psoriasis-like skin lesions. These lesions were characterized by inflammation, hyperproliferation, and abnormal differentiation of keratinocytes as well as by disruption of the actin cytoskeleton. Ultrastructural analysis revealed markedly reduced cell-cell and cell-matrix contacts and loss of cell compaction in all epidermal layers. siRNA-mediated knockdown of SRF in primary human keratinocytes revealed that the cytoskeletal abnormalities and adhesion defects were a direct consequence of the loss of SRF. In contrast, the hyperproliferation observed in vivo was an indirect effect that was most likely a consequence of the inflammation. These results reveal that loss of SRF disrupts epidermal homeostasis and strongly suggest its involvement in the pathogenesis of hyperproliferative skin diseases, including psoriasis.

The knock-out of ARP3a gene affects F-actin cytoskeleton organization altering cellular tip growth, morphology and development in moss Physcomitrella patens.

The seven subunit Arp2/3 complex is a highly conserved nucleation factor of actin microfilaments. We have isolated the genomic sequence encoding a putative Arp3a protein of the moss Physcomitrella patens. The disruption of this ARP3A gene by allele replacement has generated loss-of-function mutants displaying a complex developmental phenotype. The loss-of function of ARP3A gene results in shortened, almost cubic chloronemal cells displaying affected tip growth and lacking differentiation to caulonemal cells. In moss arp3a mutants, buds differentiate directly from chloronemata to form stunted leafy shoots having differentiated leaves similar to wild type. Yet, rhizoids never differentiate from stem epidermal cells. To characterize the F-actin organization in the arp3a-mutated cells, we disrupted ARP3A gene in the previously described HGT1 strain expressing conditionally the GFP-talin marker. In vivo observation of the F-actin cytoskeleton during P. patens development demonstrated that loss-of-function of Arp3a is associated with the disappearance of specific F-actin cortical structures associated with the establishment of localized cellular growth domains. Finally, we show that constitutive expression of the P. patens Arp3a and its Arabidopsis thaliana orthologs efficiently complement the mutated phenotype indicating a high degree of evolutionary conservation of the Arp3 function in land plants.

Blood-Borne Circadian Signal Stimulates Daily Oscillations in Actin Dynamics and SRF Activity.

In peripheral tissues circadian gene expression can be driven either by local oscillators or by cyclic systemic cues controlled by the master clock in the brain's suprachiasmatic nucleus. In the latter case, systemic signals can activate immediate early transcription factors (IETFs) and thereby control rhythmic transcription. In order to identify IETFs induced by diurnal blood-borne signals, we developed an unbiased experimental strategy, dubbed Synthetic TAndem Repeat PROMoter (STAR-PROM) screening. This technique relies on the observation that most transcription factor binding sites exist at a relatively high frequency in random DNA sequences. Using STAR-PROM we identified serum response factor (SRF) as an IETF responding to oscillating signaling proteins present in human and rodent sera. Our data suggest that in mouse liver SRF is regulated via dramatic diurnal changes of actin dynamics, leading to the rhythmic translocation of the SRF coactivator Myocardin-related transcription factor-B (MRTF-B) into the nucleus.

Recruitment of Matrix Metalloproteinase-9 (MMP-9) to the Fibroblast Cell Surface by Lysyl Hydroxylase 3 (LH3) Triggers Transforming Growth Factor-β (TGF-β) Activation and Fibroblast Differentiation.

Solid tumor growth triggers a wound healing response. Similar to wound healing, fibroblasts in the tumor stroma differentiate into myofibroblasts (also referred to as cancer-associated fibroblasts) primarily, but not exclusively, in response to transforming growth factor-β (TGF-β). Myofibroblasts in turn enhance tumor progression by remodeling the stroma. Among proteases implicated in stroma remodeling, matrix metalloproteinases (MMPs), including MMP-9, play a prominent role. Recent evidence indicates that MMP-9 recruitment to the tumor cell surface enhances tumor growth and invasion. In the present work, we addressed the potential relevance of MMP-9 recruitment to and activity at the surface of fibroblasts. We show that recruitment of MMP-9 to the fibroblast cell surface occurs through its fibronectin-like (FN) domain and that the molecule responsible for the recruitment is lysyl hydroxylase 3 (LH3). Functional assays suggest that both pro- and active MMP-9 trigger α-smooth muscle actin expression in cultured fibroblasts, reflecting myofibroblast differentiation, possibly as a result of TGF-β activation. Moreover, the recombinant FN domain inhibited both MMP-9-induced TGF-β activation and α-smooth muscle actin expression by displacing MMP-9 from the fibroblast cell surface. Together our results uncover LH3 as a new docking receptor of MMP-9 on the fibroblast cell surface and demonstrate that the MMP-9 FN domain is essential for the interaction. They also show that the recombinant FN domain inhibits MMP-9-induced TGF-β activation and fibroblast differentiation, providing a potentially attractive therapeutic reagent toward attenuating tumor progression where MMP-9 activity is strongly implicated.

Histone demethylase retinoblastoma binding protein 2 regulates the expression of α-smooth muscle actin and vimentin in cirrhotic livers

Liver cirrhosis is one of the most common diseases of Chinese patients. Herein, we report the high expression of a newly identified histone 3 lysine 4 demethylase, retinoblastoma binding protein 2 (RBP2), and its role in liver cirrhosis in humans. The siRNA knockdown of RBP2 expression in hepatic stellate cells (HSCs) reduced levels of α-smooth muscle actin (α-SMA) and vimentin and decreased the proliferation of HSCs; and overexpression of RBP2 increased α-SMA and vimentin levels. Treatment with transforming growth factor β (TGF-β) upregulated the expression of RBP2, α-SMA, and vimentin, and the siRNA knockdown of RBP2 expression attenuated TGF-β-mediated upregulation of α-SMA and vimentin expression and HSC proliferation. Furthermore, RBP2 was highly expressed in cirrhotic rat livers. Therefore, RBP2 may participate in the pathogenesis of liver cirrhosis by regulating the expression of α-SMA and vimentin. RBP2 may be a useful marker for the diagnosis and treatment of liver cirrhosis.

Anti-lipopolysaccharide factor and crustin-III, the anti-white spot virus peptides in Penaeus monodon: Control of viral infection by up-regulation

White Spot Syndrome Virus (WSSV) is the most devastating disease affecting shrimp culture around the world. Though, considerable progress has been made in the detection and molecular characterization of WSSV in recent years, information pertaining to immune gene expression in shrimps with respect to WSSV infection remains limited. In this context, the present study was undertaken to understand the differential expression of antimicrobial peptide (AMP) genes in the haemocytes of Penaeus monodon in response to WSSV infection on a time-course basis employing semi-quantitative RT-PCR. The present work analyzes the expression profile of six AMP genes (ALF, crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5), eight WSSV genes (DNA polymerase, endonuclease, immediate early gene, latency related gene, protein kinase, ribonucleotide reductase, thymidine kinase and VP28) and three control genes (18S rRNA, β-actin and ELF) in P. monodon in response to WSSV challenge. Penaeidins were found to be up-regulated during early hours of infection and crustin-3 during late period of infection. However, ALF was found to be up-regulated early to late period of WSSV infection. The present study suggests that AMPs viz. ALF and crustin-3 play an important role in antiviral defense in shrimps. WSSV gene transcripts were detected post-challenge day 1 itself and increased considerably day 5 onwards. Evaluation of the control genes confirmed ELF as the most reliable control gene followed by 18S rRNA and β-actin for gene expression studies in shrimps. This study indicated the role of AMPs in the protection of shrimps against viral infection and their possible control through the up-regulation of AMPs

Glypican-3 regulates migration, adhesion and actin cytoskeleton organization in mammary tumor cells through Wnt signaling modulation

Glypican-3 (GPC3) is a proteoglycan involved in migration, proliferation and cell survival modulation in several tissues. There are many reports demonstrating a downregulation of GPC3 expression in some human tumors, including mesothelioma, ovarian and breast cancer. Previously, we determined that GPC3 reexpression in the murine mammary adenocarcinoma LM3 cells induced an impairment of their in vivo invasive and metastatic capacities together with a higher susceptibility to in vitro apoptosis. Currently, the signaling mechanism of GPC3 is not clear. First, it was speculated that GPC3 regulates the insulin-like growth factor (IGF) signaling system. This hypothesis, however, has been strongly challenged. Recently, several reports indicated that at least in some cell types GPC3 serves as a selective regulator of Wnt signaling. Here we provide new data demonstrating that GPC3 regulates Wnt pathway in the metastatic adenocarcinoma mammary LM3 cell line. We found that GPC3 is able to inhibit canonical Wnt signals involved in cell proliferation and survival, as well as it is able to activate non canonical pathway, which directs cell morphology and migration. This is the first report indicating that breast tumor cell malignant properties can be reverted, at least in part, by GPC3 modulation of Wnt signaling. Our results are consistent with the potential role of GPC3 as a metastasis suppressor.

Tumor Necrosis Factor alpha and Interleukin-1 beta Modulate Calcium and Nitric Oxide Signaling in Mechanically Stimulated Osteocytes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Stem cell factor estimula células da musculatura lisa da traqueia a produzir TGF-β, FGF-2 E CCL3/MIP-1α

Pós-graduação em Ciências Fisiológicas - FOA

Increased fibroblast telomerase expression precedes myofibroblast alpha-smooth muscle actin expression in idiopathic pulmonary fibrosis

OBJECTIVE: This study sought to identify the relationship between fibroblast telomerase expression, myofibroblasts, and telomerase-mediated regulatory signals in idiopathic pulmonary fibrosis. METHODS: Thirty-four surgical lung biopsies, which had been obtained from patients with idiopathic pulmonary fibrosis and histologically classified as usual interstitial pneumonia, were examined. Immunohistochemistry was used to evaluate fibroblast telomerase expression, myofibroblast alpha-smooth muscle actin expression and the tissue expression of interleukin-4, transforming growth factor-beta, and basic fibroblast growth factor. The point-counting technique was used to quantify the expression of these markers in unaffected, collapsed, mural fibrosis, and honeycombing areas. The results were correlated to patient survival. RESULTS: Fibroblast telomerase expression and basic fibroblast growth factor tissue expression were higher in collapsed areas, whereas myofibroblast expression and interleukine-4 tissue expression were higher in areas of mural fibrosis. Transforming growth factor-beta expression was higher in collapsed, mural fibrosis and honeycombing areas in comparison to unaffected areas. Positive correlations were found between basic fibroblast growth factor tissue expression and fibroblast telomerase expression and between interleukin-4 tissue expression and myofibroblast alpha-smooth muscle actin expression. Negative correlations were observed between interleukin-4 expression and basic fibroblast growth factor tissue expression in areas of mural fibrosis. Myofibroblast alpha-smooth muscle actin expression and interleukin-4 tissue expression in areas of mural fibrosis were negatively associated with patient survival. CONCLUSION: Fibroblast telomerase expression is higher in areas of early remodeling in lung tissues demonstrating typical interstitial pneumonia, whereas myofibroblast alpha-smooth muscle actin expression predominates in areas of late remodeling. These events seem to be regulated by basic fibroblast growth factor and interleukin-4 tissue expression, respectively.