Lucia Schuger - US grants

Lung development is a continuous process involving epithelio-mesenchymal interactions. Laminin, a basement membrane-related glycoprotein, plays a critical role in this process. Preliminary evidence indicates that both epithelium and mesenchyme produce laminin, and that the biological activity of this glycoprotein in the mouse embryonic lung is mediated at least by two different molecular domains. The goal of these investigations will be to precisely define laminin sites active during murine lung development and to establish structure-function correlations. The effects of a series of antibodies to specific laminin epitopes, synthetic peptides, including sequences from the two molecular domains of interest, and laminin fragments will be used for functional studies. Cell-cell and cell-basement membrane interactions will be perturbed by microinjecting nanomolar quantities of these probes into the extracellular space of the epithelial and mesenchymal compartments of embryonic lung explants developing in vitro. The effects of these interventions on morphogenesis, branching activity (at branching and non-branching points), cell phenotype, proliferation and basement membrane formation will be determined. These studies will establish whether the effects of laminin in the lung are general or cell/site- specific and will define molecular sites biologically active in lung organogenesis. The role of laminin active sites in modulating primary cell behaviors essential for morphogenesis will be established by using synthetic peptides and antibodies to promote or block epithelial and mesenchymal cell adhesion, motility and proliferation. The role of laminin in morphogenic events requiring cell-cell recognition will be elucidated next by determining molecular domains involved in cell sorting and polarization as it takes place in organotypic cultures of embryonic lung cells. Elucidation of laminin involvement in adhesion, motility, proliferation, sorting and polarization of epithelial and mesenchymal cells will provide the cellular basis to further interpret the results observed at the organ culture level. Mechanistic conclusions will be drawn on how epithelium, mesenchyme and laminin interact during normal lung development. Eventually, this investigation will define possible reciprocal interactions between epithelium and mesenchyme in the control of laminin expression by studying the effects of transient exposure of one cell type to the other. A combination of SDS-PAGE, Northern blot analysis, and mRNA in situ hybridization will be employed to detect changes in laminin expression. This integrative approach will provide new information important for the understanding of normal lung development. It may also contribute to the elucidation of pathological conditions that result or lead to defective lung development, such as lung hypoplasia, bronchopulmonary sequestration, bronchopulmonary dysplasia, as well as other abnormal conditions in which the extracellular matrix is involved.

Lung development is a continuous process involving epithelial-mesenchymal interactions. The long term goal of our research is to understand the role of laminins in this process. Laminins are basement membrane-related glycoproteins produced by epithelial and mesenchymal cells and best characterized by their involvement in cell adhesion, migration and maintenance of the differentiated cell phenotype. Cells bind to laminins in part via the integrin family of extracellular matrix receptors. These are heterodimeric transmembrane glycoproteins with roles in signal transduction. During the current funding period we have found that different domains of laminins -1 and -2 are engaged in promoting lung morphogenesis by serving different functions; among them, the production of a polymer at the epithelial-mesenchymal interface seems critical for the maintenance of normal tissue structure. Furthermore, discontinuities in this laminin polymer result in direct epithelial-mesenchymal contact. Based on our recent studies we hypothesize that such heterotypic cell-cell contact induces the expression of laminins - 1 and -2 and their deposition at the epithelial-mesenchymal interface. The process is initiated by an integrin-mediated mechanism and results in basement membrane formation, differentiation of peribronchial mesenchyme into smooth muscle and budding of the bronchial tree. The specific aims of this proposal are designed to test this hypothesis. During this investigation we shall: 1. characterize the mechanism involved in the secretion of laminin-2 to the extracellular compartment upon epithelial-mesenchymal contact and the role of integrins in this process; 2. determine the mechanism of regulation of laminin-1 expression in epithelial-mesenchymal cocultures; 3. elucidate the role of laminin-1 in bronchial smooth muscle cell differentiation and identify specific laminin-1 domains involved in the process; and 4. determine the role of laminin polymerization/depolymerization at the epithelial-mesenchymal interface in epithelial budding and in mesenchymal paracrine control over epithelium. We anticipate that each of the four aims proposed here will provide new insight in the process of organogenesis and contribute to the elucidation of pathological conditions that result or lead to defective lung development.

Bronchial myogenesis involves the differentiation of local embryonic mesenchymal cells into smooth muscle (SM) cells. The mechanisms that determine this process are largely unknown. Our recent studies suggested that laminin-2 induces SM differentiation by promoting mesenchymal cell spreading/elongation ( 1-3). In unrelated studies we found that heterogeneous ribonucleoprotein-H (hnRNP-H), an RNA-binding protein involved in alternative splicing, inhibits SM differentiation and bronchial myogenesis. Based on our preliminary data, we hypothesize that bronchial myogenesis involves activation of a laminin-integrin mediated pathway that results in downregulation of hnRNP-H. This in turn causes a switch in serum response factor (SRF) pre-mRNA alternative splicing that favors SM gene expression. We also hypothesize that altered hnRNP-H levels contribute to the myofibroblast phenotype seen in lung disease. The studies proposed in this application will address these hypotheses. Aim #1 will elucidate whether stimulation of SM differentiation by laminin-2 involves regulation of hnRNP-H and what is the signaling pathway connecting them. Based on preliminary studies we will focus on integrins alpha1beta1 and alpha2beta1, RhoA and MAPK 38. Aim #2 will establish the role of hnRNP-H in SRF pre-mRNA alternative splicing and how each SRF isoform impacts on hnRNP-H-mediated SM differentiation. It has been recently shown that SRF pre-mRNA splices into dominant positive and negative isoforms and our preliminary data suggest that these are involved in bronchial myogenesis. Aim #3 will determine whether modulation of hnRNP-H levels/activity affects the phenotype of mature SM cells, fibroblasts and lung myofibroblasts. The myofibroblasts will be studied using a bleomycin model of lung fibrosis. Our pilot data showed a decrease in hnRNP-H levels in this model. In summary, this project will define a novel molecular pathway controlling bronchial myogenesis. This pathway may be shared by many different myogenic stimuli. We anticipate that these studies will provide novel knowledge that will advance considerably our understanding of lung development and diseases caused by, or resulting from abnormal SM quantity, distribution and function.

DESCRIPTION (provided by applicant): Cell elongation, either due to spread or stretch, induces lung embryonic mesenchymal cells to follow a myogenic pathway [1-3]. Using these systems we identified by suppressive subtraction hybridization (SSH) two similar stretch-responsive factors, which we referred to as tension-induced/inhibited proteins (TIPs) -1 and -3. A third isoform of intermediate Mf/, TIP-2, was identified in the GenBank, hut it was not present in the lung. A Genomic BLAST search indicated that the three TIPs were originated by alternative splicing from a single gene. Immunohistochemical studies and tagging with green fluorescence protein (GFP) demonstrated that TIPs translocate between nucleus and cytoplasm. TIPs display signature motifs characteristic of nuclear receptor coregulators and chromatin remodeling enzymes. Functional studies encompassing overexpression of TIPs and gene silencing using small interfering RNAs (siRNA) indicated that TIP-1 and -3 are involved in the cell's selection between the myogenic and adipogenic pathways. TIP- 1, induced by stretch, promotes myogenesis while TIP-3, inhibited by tension initiates adipogenesis. Furthermore, chromatin immunoprecipitation (ChIP) assays indicated that TIPs may act through a mechanism of chromatin remodeling. Since the lung has bronchial smooth muscle (SM), interstitial myofibroblasts and lipofibroblasts, we hypothesize that the development of these cell types is determined at least in part by TIPs. The specific aims of this project will be: Aim 1. To determine whether modulation of TIP-1 and TIPS alters bronchial SM, myofibroblasts and lipofibroblasts development in lung organ cultures and organotypic cultures. Aim 2. To elucidate the transcription factors that are activated by TIP-1 and TIP-3 and whether gene acetylation, methylation or both are involved in TIP-induced transcription activation. Aim 3. To identify the histone(s) that bind(s) to TIPs and to determine the TIP motifs that are required for histone-TIP binding. Bronchial and vascular SM, myofibroblasts and lipofibroblasts are essential components of the lung. These cells participate in multiple physiological processes and however very little is known about their development and the molecular mechanisms involved in their genesis. The studies proposed in this application will provide therefore novel information on how the myogenic and adipogenic fates are determined in the embryonic lung at the cellular and molecular level.

Pulmonary lymphangioleiomyomatosis (LAM) is a poorly understood disorder characterized by abnormal smooth muscle (SM)-like, tuberin-minus cell proliferation leading to lung destruction and pseudocyst formation. Serum response factor (SRF) is a transcription factor that plays an essential role in the regulation of SM myogenesis and cell growth. We recently found that LAM lesions exhibit high SRF expression levels. Furthermore, our studies demonstrated that upregulation of SRF in normal lung fibroblasts stimulates production of key extracellular matrix (ECM)-degrading enzymes including several metalloproteinases (MMPs) and urokinase plasminogen activator (uPA). On the contrary, their corresponding inhibitors, tissue inhibitor of metalloproteinases-3 (TIMP-3) and plasminogen activator inhibitor-1 (PAI-1) are downregulated, resulting in an enzymatic imbalance that favors ECM degradation. Immunohistochemistry and laser microcapture confirmed that this imbalance is also present in LAM lesions. Since SRF is in addition a well established mitogenic transcription factor we hypothesize that high SRF levels contribute to the pathogenesis of LAM by creating a pro-proteolvtic imbalance that favors ECM degradation and LAM cell proliferation/survival. Hence, SRF downregulation should eliminate these deleterious features. Furthermore, we hypothesize that exogenous TIMP-3 should also correct the proteolvtic imbalance in a more direct manner. To address our hypotheses we developed unique LAM culture systems, including LAM cell primary cultures (identifying LAM cells based on two novel LAM cell markers), lung explant cultures, and LAM xenografts in SCID mice. Here we specifically propose: 1- To determine whether SRFdownregulation will correct the pro-proteolytic imbalance in LAM cells. 2- To elucidate whether SRF downregulation will change LAM cell behavior with regards to ECM invasion and cell proliferation/apoptosis. 3- To determine whether exogenous TIMP-3 will decrease the activity of MMPs and improve LAM cell behavior. Our hypotheses will also be tested on ELT-3, a tuberin-minus cell line, to integrate the proposed studies with existing research on LAM. We believe that the new knowledge obtained from these studies will help to better understand the pathogenesis of LAM and to devise novel therapeutic strategies to treat this disease.

DESCRIPTION (provided by applicant): P311 is an 8 kDa intracellular protein highly conserved across species. P311 does not belong to any established protein family;neither has signature motifs that could suggest function. In mouse and human P311 is produced in the brain, in vascular smooth muscle [11, 24], in wound myofibroblasts [11] and their precursors, the pre-myofibroblasts [11];and in blood monocytes (research plan, figures 5 and 7). By performing telemetry studies we recently found that p311-deficient mice (P311 KOs) have pronounced systemic systolic and diastolic hypotension, without overt gross or microscopic vascular abnormalities. Echocardiographic, morphometric and myosin light chain 20 (MLC20) phosphorylation studies indicated that the hypotension is due to a decrease in vascular tone rather than the result of a vascular structural abnormality. Hypotension is accompanied by a significant downregulation in vascular TGF-[unreadable]1 level and activity, which occurs at the translation and/or degradation stage. More importantly, P311 KOs were significantly protected against the development of uninephrectomy-DOCA-induced hypertension. Pertinent to the Challenge Topic selected, additional studies showed a stable and tight correlation between vascular and monocyte P311 expression but a significant difference in P311 expression was observed between mice with different genetic background, such that CD-1 mice express over 2-fold less P311 than the BL/6 strain. Significant differences in monocyte P311 expression were also observed between human healthy volunteers. Since lack of P311 ameliorated experimentally-induced hypertension, we compared the hypertensive response in mice with low (CD-1) and high (BL/6) monocytic P311 level. This preliminary study showed that while both groups were normotensive under baseline conditions, the mice with low P311 developed significantly less severe hypertension than the mice with high P311 level (p<0.0001). Based on these preliminary observations we hypothesize that P311 plays an important role in the modulation of systemic blood pressure and that the level of P311 in peripheral blood monocytes is a useful biomarker to predict susceptibility for the development and/or severity of future hypertension. To test these hypotheses, the specific aims of this proposal are: 1. To determine whether P311 expression level and its vascular-monocyte correlation, observed in preliminary studies, change according to age and sex. 2. To determine whether the baseline level of monocyte/vascular P311 expression correlates with the severity of experimentally-induced hypertension, as suggested by preliminary studies. 3. To determine whether vascular P311 overexpression causes hypertension and/or increases the severity of experimentally-induced hypertension. The experiments proposed in aim #1 will demonstrate whether P311 level of expression in blood vessels and monocytes depends on the mice genetic background, whether it changes over time in male and female mice of same genetic background and whether its vascular level can be accurately assessed by determining P311 mRNA level in peripheral blood monocytes. The full scale experiments proposed in aim #2 and part of aim #3 will hopefully validate and expand our preliminary studies by unequivocally demonstrating that the basal P311 expression level in mice directly correlates with the hypertensive response in an experimental model of induced hypertension. If the studies proposed in aim #1 and #2 yield the expected outcomes, we could conclude that P311 expression level in monocytes can be accurately determined and represents a reliable predictor of hypertension susceptibility in mice. In such a case, the clinical usefulness of P311 as a biomarker for human susceptibility to hypertension should warrant direct exploration. Finally, the experiments proposed in aim #3 will demonstrate whether, at least in mice, high P311 levels can play a causative/contributory role in hypertension. In such a case, P311 could represent an additional target for the development of novel therapeutic approaches. PUBLIC HEALTH RELEVANCE: P311 is an 8 kDa intracellular protein of unknown function. In humans and mice P311 is produced by certain neurons, by vascular smooth muscle and by monocytes (a type of white blood cell). Based on preliminary studies, the main goal of this project is to demonstrate that baseline P311 level in blood monocytes represents a useful biomarker to predict the future development and/or severity of hypertension in mice.

DESCRIPTION (provided by applicant): Hypertrophic scars (HSs) occur at a high incidence after burns and lack effective treatment. TGF-?, a powerful profibrogenic growth factor, plays an important role in HS development. We recently demonstrated that P311, an 8 kDa intracellular protein present in human HSs, controls TGF-?1-3 level/activity by stimulating their translation, and preliminary work suggested that P311 directly binds to eukaryotic translation initiation factor 3b (eIF3b). We developed a murine model of HS by topical application of bleomycin (BLM) on skin excisional wounds. P311 is expressed in these HSs, but not in normal scars. Pilot studies suggested that P311 KO mice are protected against BLM-induced HSs, while P311 transgenic mice (P311 TGs) develop HSs in the absence of BLM. P311 has a conserved, PEST-like motif, known to mediate protein-protein interactions. A peptide representing the P311 PEST-like motif or a P311 siRNA, each of them conjugated to the cellular penetrating peptide TAT47-57 (TAT47-57P311PEST and TAT47-57P311siRNA), targeted P311 and reduced TGF-?s levels in vitro, moreover, the TAT47-57P311siRNA ameliorated BLM-induced HSs. Based on the above, we hypothesize that P311 promotes HSs by binding to eIF3b and thereby stimulating TGF-?s translation; and that topical application of TAT47-57P311PEST or TAT47-57P311siRNA will protect mice from developing HSs. The specific aims to test this hypothesis are: 1. To determine the involvement of P311 in mouse HSs and whether it is mediated by the P311 PEST-like domain. We will characterize the BLM-treated scars of P311 KO mice and WTs, the scars of P311 TGs, and the effect of P311 and P311 PEST mutants intradermally delivered at the wound site. The read-outs will include histological quantification of fibrosis using the Aperio system pls immunoblot/densitometry; quantification of myofibroblasts; and determination of P311 and TGF-?1-3 mRNAs and protein levels. Mouse and human keratinocyte (KT) and dermal fibroblast (DF) response to P311 will be studied in primary tissue cultures 2. To determine the effect of P311 and its PEST-like domain in TGF-?s translation by mouse and human keratinocytes (KTs) and dermal fibroblasts (DFs), and whether it is mediated by P311 interaction with eIF3b. We will employ KT and DF primary cultures, P311 and P311 PEST mutants delivered by lentivirus transfer, and purified P311 and eIF3b. Among the read outs will be TGF-? 1-3 translation reporter assays, co-immunoprecipitations, protein pull-downs, use of surface plasmon resonance, RNA immunoprecipitation assays and TGF-?1-3 translation reporter assays after eIF3b knockdown by RNA interference. 3. To determine whether topically-delivered TAT47-57P311PEST and TAT47-57P311siRNA are effective in preventing BLM-induced HSs. Protocols for the topical delivery of each of these two compounds in P311 TG mice wounds/scars and BLM-treated wounds/scars will be optimized and their effect in preventing the development of HSs will be determined/compared. Read-outs will be the same as for aim #1.

ABSTRACT Interstitial lung fibrosis is more frequently idiopathic and fatal. TGF-? is one of the main players in idiopathic pulmonary fibrosis (IPF). P311 is a highly conserved RNA binding protein that stimulates TGF-?s 1-3 translation through its interaction with eukaryotic translation initiation factor 3b. P311 is expressed in alveolar epithelial cells (AEC) and mesenchymal cells (MCs) in IPF and in bleomycin (BLM)-induced pulmonary fibrosis in mice, but not in normal lung. Our studies indicated that P311 knock out mice (P311 KOs) are highly protected against BLM-induced pulmonary fibrosis and have lower TGF-?s 1-3 than WT controls, while P311 transgenic mice (P311 TGs) develop spontaneous lung fibrosis and have high TGF-?s 1-3. Furthermore, BLM induces P311 expression in AEC and MS cultures. In silico analysis disclosed conserved binding sites for eleven transcription factors (TFs) in the promoter region of P311 and a pilot study suggested involvement of at least one of them in BLM-induced P311 expression in AECs but not in MCs. Initial studies showed that a synthetic peptide composed of five amino acids (aas) of the P311 RNA recognition motif (RRM) and the cell penetrating peptide TAT47-57 aa sequence (P311RRM-TAT) penetrated lung AECs and downregulated TGF-?s translation. Furthermore, a P311 siRNA conjugated to TAT47-57 (P311siRNA-TAT) protected from BLM-induced lung fibrosis. Based on the above, we hypothesize that P311 induces pulmonary fibrosis by differentially acting on AECs and MCs; that it does so in part by stimulating TGF-?s translation; that factors upstream and downstream of P311, other than TGF-?s, are involved in P311-mediated fibrosis; and that intratracheal delivery of P311RRM-TAT or P311siRNA-TAT will protect mice from experimentally-induced fibrosis . The specific aims to test our hypotheses are: 1. To fully characterize the involvement of P311 in experimentally induced pulmonary fibrosis. We will perform the basic studies needed to solidify the main tenet of our working hypothesis. Acute and chronic models of lung fibrosis in wild type (WT), P311 KOs and P311 TGs and standard fibrosis read outs will be used to accomplish this aim. 2. To determine the effect of P311 on AECs and MCs responses associated with fibrosis. The read outs will include cell proliferation, death, migration, collagens production, extracellular matrix (ECM) proteinases expression, ECM proteolysis, and myofibroblast differentiation. 3. To identify upstream and downstream P311 effectors with a role in pulmonary fibrosis. The involvement of TGF-?s 1-3 as downstream P311 effectors will be determined in animal models of lung fibrosis by gain and loss of function experiments. To identify other P311 downstream effectors we will perform CLIPseq assays. To identify P311 upstream effectors we will perform loss and gain of function studies targeting the TFs suggested by the in silico analysis. The involvement of selected P311 effectors on mouse lung fibrosis will be determined. 4. To determine whether intratracheally delivered P311RRM-TAT and P311siRNA-TAT are effective in the treatment of experimentally-induced pulmonary fibrosis.

ABSTRACT Lymphangioleiomyomatosis (LAM) is a rare, low grade neoplasia affecting primarily women of childbearing age. LAM is characterized by the nodular proliferation of SMA and HMB-45 positive LAM cells throughout the lung, producing cystic destruction and respiratory insufficiency. Mutations in the tsc2 or tsc1 genes leading to activation of the mTOR pathway underlie the disease. LAM cells express mainly progesterone receptors (PRs) rather than estrogen receptors; however the role of PRs in the disease has not been investigated. Preliminary studies using a new, well characterized, sporadic-LAM-derived LAM cell line (LAM1) which expresses PRs suggested that the progesterone (P4)/PR axis stimulates LAM cell proliferation, extracellular matrix (ECM) proteinases expression, and pericellular proteolysis, all independently of mTOR activation, whereas mifepristone, a P4 antagonist, abolishes these effects. In a separate pilot study we found that TSC2 transfer suppresses PR expression in LAM1 cells. Additionally, we found that a LAM cell subpopulation expresses PLAP (placental alkaline phosphatase) and preliminary studies suggested a direct correlation between PR positivity and level of secreted PLAP. Finally, intratracheal delivery of LAM1 cells to ovariectomized immunodeficient female mice receiving P4 resulted in LAM-like features undistinguishable from human pulmonary LAM. Based on these observations we hypothesize that: A. the P4/PRs axis contributes to the pathogenesis of LAM by stimulating LAM cell malignant behavior and lung cystic destruction; B. PR expression in LAM results from lack of TSC2-mediated PR transcription repression. C. PLAP levels in serum are a marker for PR positive (PR+) LAM cell burden; and D. concomitant blockage of mTOR and the P4/PR axis is more effective in preventing/ameliorating LAM manifestations in an orthotopic mouse model that blockage of mTOR alone. To test these hypotheses we specifically propose: 1. A To determine the involvement of the P4/PR axis in the promotion of LAM cellular features associated with a low grade neoplastic behavior, including LAM1 cell proliferation, death, production of ECM proteolytic enzymes, etc. 1. B To determine the involvement of the P4/PR axis in the development of pulmonary LAM-like disease in an orthotopic mouse model by quantifying LAM cell burden, cystic destruction, and metastatic dissemination. 1. C To determine the effect of the P4 antagonists mifepristone and lonaprisan on LAM cell low grade neoplastic behavior in vitro and in vivo using the same read outs presented in sub-aims A and B. 2. To determine the effect of TSC2 transfer on pr transcription by LAM1 cells using qPCR, promoter reporter assays and ChIP analysis. 3. To assess whether PLAP level is a marker of PR+ LAM cell burden by determining the potential in vitro and in vivo correlation between PR+ LAM cell burden and PLAP levels in medium and serum. 4. To compare the effectivity of rapamycin alone versus rapamycin plus a P4 antagonist in the treatment of LAM-like disease in an orthotopic mouse model. The experimental protocol will be modeled on that presented in aim 1 B, with same read outs.