Rationale for the Study of

Binding 5 in Alveolar Type II Cells

Derek Garrison

Master of Science Thesis

Molecular and Developmental Biology Graduate Program

University of Cincinnati College of Medicine Advisor: Jeff Whitsett, MD

Autumn 2008

Premature birth is currently the leading cause of infant morbidity and mortality in the United States.

One common aspect of premature birth leading to morbidity and mortality is lung immaturity, which often entails a lack of proper lung morphogenesis and pulmonary surfactant production (1,2). Presently, a thorough understanding of the molecular regulation, transport, and modification of lipids required to produce functional pulmonary surfactant is lacking, which hinders progress toward improved care for patients with immature lungs. Understanding the molecular mechanisms regulating lipid trafficking will allow the development of more effective preventative and treatment strategies for patients with immature lung pathologies like Respiratory Distress Syndrome (RDS). The following document describes the rationale for the study of Fatty acid binding protein 5 (FABP5) in alveolar type II cells. Previous deletion and over- expression data implicate Fabp5 as being a potential component of lipid regulation during perinatal lung maturation. Specifically, Fabp5 down-regulation strongly correlates with decreased lipogenesis and improper production of pulmonary surfactant. In addition, Fabp5 expression has been shown to be regulated by known to control lipogenesis in vivo . As a result, this paper describes the previous and bioinformatic data supporting the hypothesis that Fabp5 regulates lung lipid homeostasis.

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Acknowledgments

I would personally like to thank the members of the Whitsett lab, Molecular and Developmental

Biology program faculty, staff, and students, and other division colleagues for their support in completing this degree. Most importantly, I would like to thank my wife, Sara, and family for being supportive and empowering throughout this endeavor.

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Table of Contents

i. Introduction

ii. Background

iii. Surfactant Regulation

iv. FABP Background

v. FABP Regulation

vi. FABP5 Background

vii. Proliferation viii. Lipid-Binding Functions

ix. Regulation

x. Lung Transcriptional Regulation

xi. Compensation and TII Cell Function

xii. Conclusion

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Introduction

Premature birth is currently the leading cause of infant morbidity and mortality in the United States

(U.S. Dept. of Health and Services, 2006). One common aspect of premature birth leading to morbidity and mortality is lung immaturity, which often entails a lack of proper lung morphogenesis and pulmonary surfactant production (1,2). Pulmonary surfactant is necessary for lung function during aerobic respiration, and improper surfactant homeostasis in the lung is known to lead to Respiratory Distress

Syndrome (RDS) and other acute and chronic pathologies in adults and infants (3,4). Current treatments for lung immaturity, such as high oxygen, continuous positive airway pressure (CPAP), and hormonal therapies do not eliminate chronic lung injuries (5,6). Presently, a thorough understanding of the molecular regulation, transport, and modification of lipids required to produce functional pulmonary surfactant is lacking, which hinders progress toward improved care for patients with immature lungs. Therefore, this proposal seeks to present the theoretical rationale for studying the potential role of Fatty acid binding protein 5 (FABP5) during perinatal lung lipid regulation. Understanding the molecular mechanisms regulating lipid trafficking will allow the development of more effective preventative and treatment strategies for patients with immature lung pathologies like RDS.

Background

Pulmonary surfactant is necessary to maintain proper surface tension and alveolar sterility in the lung. Lipids represent 90% of the pulmonary surfactant pool, with phosphatidylcholine (PC) being the most highly expressed lipid and cholesterol the most prevalent neutral lipid (1). The other 10% of surfactant is composed of the four surfactant proteins: Surfactant proteins A, B, C, and D (1,2). Surfactant lipids and proteins are processed and organized into lamellar bodies in alveolar type II (TII) cells (1). Lamellar bodies are surfactant storage vesicles that fuse with the apical plasma membrane of TII cells leading to alveolar surfactant secretion (7). Alteration of the lipid and protein components of lamellar body and pulmonary surfactant production in TII cells leads to RDS (3,8). During late gestation the distal lung begins accumulating lipids for incorporation into the surfactant before its secretion just prior to parturition (9,10).

These lipids are primarily found in TII cells and lipofibroblasts (11). Concurrently, an up-regulation in the 5

expression of surfactant and lipid biosynthetic, transport, and modifying proteins is observed in TII cells

(9,10,12). The transport of lipids from the lipofibroblasts to TII cells is poorly described. Since there is no known mechanism for the transport of triglycerides (TG) across membranes, the TGs are thought to be hydrolyzed and transported as fatty acids (FA). Before FA modification in TII cells, the FAs must be stabilized and protected in order to be available for incorporation into surfactant. After lipid modification and incorporation into the LB, surfactant is secreted into the alveolus. These processes must be at least partly regulated in TII cells since lipids from both lipofibroblasts and TII cells are components of the functional surfactant pool. After the initial secretion of surfactant during late gestation and early postnatal life, the maintenance of the surfactant pool is predominately performed by TII cells. This maintenance entails both modification of adsorbed surfactant lipids and biosynthesis of new phospholipids (1). However, the regulation of the transport pathway and the proteins involved, and intersection points of the transport, uptake, and lipogenic pathways are poorly understood. This poor understanding warrants more analysis to better elucidate the molecular program regulating perinatal lipid homeostasis. Pulmonary Biology Fabp5 Decrease Fabp Increase Database Differential Expression Cebpa -2.8 Fabp4 Surfactant Regulation b1 deletion -1.47 Fabp4

Intracellular lipid content MIA misexpression -1.74 Fabp4, Fabp7 in TII cells is likely regulated at SREBP Cleavage Activating Protein deletion -1.75 -

Stat3 deletion -2.34 - multiple levels, due to its Hif deletion -1.70 Fabp7 necessary roles in metabolism, Foxa2 deletion -2.08 Fabp4, Fabp3 signaling, proliferation, and Fgfr2IIIb deletion -1.36 Fabp1, Fabp3 surfactant biosynthesis. One FGF_18 misexpression -2.08 Fabp7 level is transcriptional Figure 1. Fabp Differential Expression . Lung-specific mRNA microarray analysis of Fabp gene expression in multiple transgenic or drug-treated regulation. This lab and others animals. have previously shown that conditional loss of CCAAT/enhancer binding protein alpha (Cebp α ), Sterol regulatory element binding protein cleavage activating protein (Scap), Calcineurin b1 ( Cnb1 ), Signal transducer and activator of transcription 3 (Stat3 ), Hypoxia inducible factor 1 ( Hif1 ), and Forkhead box A2

(Foxa2) leads to deficient surfactant pools, abnormal lipid production and transport, improper lung 6

morphogenesis, and RDS after parturition (Besnard, unpublished results ) (Shannon, unpublished results )

(13-17). Each of these encodes a that has a central role in regulating the expression of multiple genes important for proper proliferation, differentiation, lipogenesis, and surfactant production. Each of the above models of lung immaturity or improper development has a significant down- regulation in the expression of Fabp5 (Figure 1). These data strongly imply that Fabp5 is a downstream target of multiple transcription factors known to regulate lipogenesis and lung maturation, suggesting that

Fabp5 is an important component of normal lipid homeostasis and lung maturation.

FABP Background

In mammals the Fatty acid binding consists of nine plasma membrane-bound and cytosolic members involved in the uptake, secretion, and intracellular transport of lipids (18). FABPs possess a characteristic tertiary structure of two orthogonal beta-barrels and a basic helix-loop-helix domain adjacent to the binding site (19). Each member binds one lipid molecule, with the exception of FABP1, which can bind two lipids simultaneously (20). FABP members possess a partially Figure 2. Conserved FABP members’ domains . There is a conserved fingerprint for all fatty acid-binding proteins (FABPs) conserved lipid binding domain across derived from three motifs. Motif 1 includes the G xW triplet, which is similar to a motif observed in . Motif 2 is located three separate motifs, and are on strand 4 ( βD) and includes strand 5 ( βE). Motif 3 encodes the last two beta-strands ( βI) and ( βJ). From Furuhashi, et al. Nat moderately conserved overall, with Rev Drug Discov., 2008. some possessing greater than 60% amino acid primary sequence similarity ( Fabp4 and Fabp3 ), and others less than 20% ( Fabp1 and Fabp5 )

(18,21) (Figure 2). 7

FABPs have been shown to be involved in lipid regulation throughout the entire body (22,23). They can be divided into three groups, depending on their ligand binding properties. Group I members, FABP1 and FABP6, can bind cholesterol, , bile salts, and bulky FAs. Group II members, FABP3, FABP4,

FABP5, FABP7, and FABP 8 bind FAs, eicosanoids, and retinoids to varying degrees. Finally, Group III’s member, FABP2, only binds FAs (18).

FABPs are targeted to the plasma membrane where they bind lipids, and are thought to then transport the lipids to a sub-cellular organelle or protein, by unknown mechanisms. FABP family members are found either uniquely or co-expressed with other FABPs in different cell types and organs (19,21,24)

(Table 1). FABP-dependent lipid transport is thought to result in regulated intracellular FA content used for protein modification, transcriptional regulation, cell metabolism, vesicular trafficking, and intracellular signaling functions by stabilizing and increasing the solubility of Table 1. FABP Gene Comparison . FABP family members’ expression profiles and chomrosomal locations across human, mouse, and rat. Furuhashi, et al. Nat Rev Drug intracellular lipids Discov., 2008. (25,26). For example, Fabp3 is known to be required for lipid uptake in cardiac cells, and its loss correlates with lower intracellular and increased extracellular fatty acid levels, leading to metabolic deficiencies (27).

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FABP Regulation

Fabp gene structure is partially conserved among all family members. Each Fabp gene possesses four exons, three introns, and a TATA box, but the intron lengths and location of the TATA box vary.

Functional promoter elements and regulatory factors appear to be unique to each FABP member’s promoter region. Jun oncogene (AP1), Hepatocyte nuclear factor 1 (HNF1), peroxisome proliferator activated (PPAR), SREBP, and Fabp Alveolar Expression

CEBP transcription factors 80000 regulate the expression of 70000 60000 multiple FABPs (18,28). For 50000 example, CEBP α inhibits the 40000 30000 AP1 activation of Fabp4 20000 Raw Signal Average expression, and Fabp7 10000 0 expression in glial cells is Fabp1 Fabp2 Fabp3 Fabp4 Fabp5 Fabp6 Fabp7 Fabp9 dependant on a POU-domain Fabp TII Expression binding site (18). Fabp9

Fabp7

Fabp6

Fabp5 FABP5 Background Fabp4

Fabp5 , also Epidermal Fabp3

Fabp , eFabp , PA-Fabp , and Fabp2 Fabp1 Mal1 , was identified from 0 10000 20000 30000 40000 50000 60000 human psoriatic skin lesions Raw Signal Average and is located on murine Figure 3. A. FABP Expression in Alveolar and TII Cells . Microarray studies of isolated and pooled four-week murine lung 3a in a 1Mb alveolar macrophages revealed Fabp1 , Fabp4 , and Fabp5 expression. B. Fabp1 , Fabp4, and Fabp5 are expressed in isolated adult TII cells region also containing Fabp4, of the distal lung.

Fabp8, and Fabp9 (29) (Table 1). Fabp5 has been found in the epidermis, macrophages, dendritic cells, retina, adipocytes, nervous system, and epithelial cells lining multiple organs, including the lung (30-42) 9

(Table 1). In the lung Fabp5 is the most highly expressed FABP in adult TII cells by mRNA (Figure 3). It is also expressed in murine and rat macrophages and lipofibroblasts (43,44). In the human lung Fabp5 is similarly found in TII cells, macrophages, Clara cells, Goblet cells, and endothelial cells (32). In Lysosomal acid lipase ( Lal ) knockout mouse lungs, Fabp5 expression is significantly increased, which is likely a result of the increased macrophage infiltration observed since Fabp5 is highly expressed in alveolar macrophages by microarray (45) (Figure 3). Fabp1 , Fabp3 , Fabp4, and Fabp7 mRNA is also expressed in the lung by microarray (Figure 1). These data suggest that Fabp5 is the major FABP in alveolar TII cells, implicating it as a potentially key component of the surfactant biosynthetic and uptake pathways.

Proliferation

Fabp5 mRNA and protein levels are up-regulated in many proliferating cell populations including cancer, during wound healing, and in immortalized epithelial cells (43,46-50).

For example, FABP5 is up-regulated in prostate cancer, and its expression positively correlates with decreased survival time (47). In addition, when PC-

3M cells are infected with Fabp5 siRNA, the cells no longer proliferate or metastasize upon implantation, implicating FABP5 as an important component regulating cellular movement and proliferation (47). The causative role of FABP5 in these Figure 4. FABP5 Binding Affinities . Purified FABP5 binding affinity was measured by PAGE/radiobinding assay for lipids processes has yet to be confirmed in listed. From Siegenthaler, et al. Biochem J, 1994. vivo .

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Lipid-Binding Functions

FABP5 plays significant roles in cell metabolism. It can bind many medium to long chain fatty acids and eicosanoids with high affinity, and squalene, cholesterol, retinoic acid, and retinol with significantly lower affinity (19,51,52) (Figure 4). A targeted null mutation at the murine FABP5 locus leads to increased insulin-induced glucose transport (53). Conversely, Fabp5 over-expression in adipocytes, utilizing the

Fabp4 enhancer, leads to increased insulin resistance and lipolysis (39,53,54). FABP5 has been shown to bind and activate Hormone sensitive lipase (HSL) and PPAR δ proteins in keratinocytes and fat cells

(26,55). This is consistent with previous data that show mammary carcinoma cells expressing high levels of

Fabp5 , when treated with RA, have increased activation of PPAR δ (56). The increased Fabp5 levels are unlikely to be due to PPAR-dependent activation since an Fabp 5 promoter element is not regulated by a

PPAR response element in HepG2 and C2C12 cells (57). These data implicate FABP5 as a key mediator of intracellular insulin and retinoic acid (RA) signaling, potentially via regulation of PPAR δ and HSL activation.

Regulation

Fabp5 is a probable target of FGFR signaling in the lung. After KGF (FGF7) addition to in vitro cultures, which functions through FGFR2IIIb, increased FABP5, dipalmitoylphosphatidylcholine (DPPC), and surfactant protein synthesis is observed in rat lungs and TII cells (43,58-60). This is supported by microarray data showing decreased Fabp5 expression after FGFR2IIIb deletion (in house) and FGF18 misexpression in the lung, both of which have improper lung morphogenesis and defective epithelial differentiation (Perl, unpublished results )(61,62) (Figure 1). KGF-dependent increase of Fabp5 expression in TII cells is not attenuated after inhibition with a dominant-negative SREBP-1c plasmid, suggesting either a SREBP-1a, SREBP-2, or SREBP-independent regulatory mechanism controls Fabp5 gene expression

(58,59) (Besnard, unpublished results ).

Fabp5 expression is up-regulated in cyclopamine-treated lungs, correlatively suggesting that the

Sonic hedgehog (Shh) pathway might negatively regulate Fabp5 expression (Shannon, unpublished results )

(Figure 1). However, SHH regulation of Fabp5 expression has not been shown in other systems, necessitating further analysis. 11

Lung Transcriptional Regulation

The mouse and human Fabp5 gene promoters are highly conserved over a 204 bp core promoter region, with conserved transcription factor binding sites (TFBS) for HOXC, Early growth response (EGR),

Myeloid 1 (MZF1), Ecotropic viral integration site 1 (EVI1), associated zinc finger (MAZ), and GC box factors SP1/GC (SP1) proteins (63-66) (Figure 5). Egr-1 is a known MAPK target, and is expressed in response to inflammation or stress in the lung after injury (67). Therefore, stress and inflammation-induced activation of the MAPK pathway could potentially regulate Fabp5 expression via Egr-1 induction. Fabp5 is a known target of MAPK pathway activation since inhibition of p38MAPK activation in

PC-12 cells inhibits the nerve growth factor-dependant increase in Fabp5 expression (68). In addition, previous data show that FABP5 partially mediates cellular inflammatory and stress responses by binding leukotriene-A4 A (eicosanoid) and Rhe 4- Hu hydroxynonenal B (lipid peroxidation cleavage product) (69,70). Figure 5. A. Conservation of Fabp5 core promoter. Comparison of the conservation MZF1, EVI1, and among multiple Fabp5 gene promoter regions reveals strong conservation with the rhesus and human promoters, and no conservation with the monkey, frog, chicken, or MAZ have poorly dog promoters. B. Conservation of Fabp5 promoter TFBS . Transcription factor binding sites conserved in the core Fabp5 mouse and human promoters, with respect to described roles in location and sequence conservation, shows only an EGR site. From Ovcharenko, et al. Nucleic Acids Res, 2004; Ovcharenko, et al. Genome Res, 2005. regulating lipogenesis and lung-specific expression. Since Fabp5 is significantly down-regulated in the lung epithelium of lung-deleted Cebp α , Scap , Cnb1 , Stat3 , and Foxa2 mice, it is of interest to see whether those transcription factors’ binding sites are found within the murine Fabp5 promoter.

In the mouse Fabp5 promoter, three cassettes containing at least one E-Box and one SREBP binding site are observed within +/- 0.5 kb of the transcriptional start site (TSS), and just 5’ of exon two 12

(Figure 6). The E-Box/SREBP element has been identified as a potential lipogenic footprint present in the promoters of multiple genes involved in lipid pathways (Xu, unpublished results ). The human Fabp5 promoter also has similar E-box/SREBP elements located at -1.0 kb upstream and +0.3 kb downstream of the TSS (Supplementary Figures 1 & 2). Also, 0.75 kb upstream and 1.0 kb downstream of the TSS there

Fabp5 Regulatory Regions A

-1.0 kb Fabp5 +1.0 kb +2.0 kb

-1.5 kb -0.5 kb +0.5 kb +1.5 kb

B -3.5 kb -2.5 kb Fabp5

-4.0 kb -3.0 kb -2.0 kb

E-BOX CEBP NKX2 SREBP PPAR STAT3 NFAT

Figure 6. A. Fabp5 Promoter Region . -1.5 kb upstream of the Fabp5 transcription start site to the beginning of the second exon. Transcription factor binding sites were obtained from Matinspector and each box represents a putative binding site for the factor associate with it. The E-Box/SREBP cassettes (red box) are putative lipid-specific regulatory regions. Other mentioned regulatory regions are circled in blue. B.. Between 2.0 and 4.0 kb 5’ to the Fabp5 transcription start site are three putative regulatory elements. Each element has an E-box other general binding sites for lipogenic program regulators (CEBP & PPAR). Of interest is that these sites are juxtaposed next to NFAT, STAT3, and NKX2 putative binding sites, which more strongly correlate these sites as being functional based on array results . are two potential regulatory cassettes containing STAT3, NKX2, CEBP, PPAR, NFAT, and E-box factor binding sites (Figure 6). There is also a potential regulatory region -2.0 to -4.0 kb upstream of the Fabp5

TSS. Within this region are three potential regulatory elements containing CEBP, PPAR, NFAT, NKX2,

STAT3, and E-Box factor binding sites (Figure 6). These potential regulatory elements should be examined when elucidating Fabp5 promoter regulation since these elements provide a potential mechanism by which the lipid or lung-specific expression or enhancement of Fabp5 could be achieved. These bioinformatics and previous transgenic mice data suggest that Fabp5 expression is regulated by factors within both the core promoter and distal regions. 13

Compensation and TII Cell Function

Previous data suggest both crosstalk and feedback regulation among Fabp genes (28,71,72). In each mouse model where Fabp5 expression is altered there exists a significant decrease or increase in another Fabp family member (Figure 1). Hepatic Fabp5 deletion leads to a compensatory increase of Fabp3 expression (71,73) . Also, deletion of Fabp4 results in increased Fabp5 expression in adipocytes (54).

These data show that Fabp5 expression both determines and is regulated by the expression levels of other

Fabp genes. However, the mechanism by which this compensation is achieved among Fabp family members is unknown. There are likely feedback regulatory events that control Fabp gene expression, but candidate promoter elements are unknown, and there is likely cell-type specific compensation as well. For example, in the lung Fabp1, Fabp3 , Fabp4 , and Fabp7 are all up-regulated in different lung immaturity models (Figure 1). In addition to promoter and compensatory mRNA analysis, functional data in multiple systems have provided insights into which pathways are upstream and downstream of Fabp5 expression and function. TII cells isolated from Fabp5 and Fabp3 double knockout mice show a decrease in labeled palmitic acid uptake and incorporation into total PC content, defect in beta-oxidation, and increased FABP1 expression (74). Palmitic acid incorporation into DPPC shows no change due to a switch from de novo synthesis to uptake and modification (74). The defects in labeled palmitic acid utilization and beta-oxidation can be rescued by PPARG stimulation, which induces Fabp1 , Caveolin-1, and Fatty acid translocase (FAT) expression (74,75). PPARG/CAVEOLIN-1 complexes have been demonstrated to interact in TII cells, providing a potential signaling complex capable of compensating for, or aiding in, activation of the lipid pathways responsible for beta-oxidation and palmitic acid incorporation into PC (74). Fabp3 and Fabp5 double deletion leads to decreased lung bronchoalveolar lavage (BAL) phospholipid/hydrophobic protein ratios and improper liquid condensed domains, which are rescued by PPARG stimulation in vitro , but not after one-week of PPARG stimulation in vivo (75). Lower lung volume is seen in the double mutant lungs, with a concurrent increase in Sp-A and Sp-D expression, but not Sp-B and Sp-C, which is of interest since both Sp-A and Sp-D are hydrophilic proteins known to play significant roles in the pulmonary surfactant recycling pathways (75-77). 14

Conclusion

Previous data and bioinformatics analysis presented above strongly suggest that Fabp5 is a potential regulator of lung lipid homeostasis. Fabp5 expression is regulated by many pathways and transcription factors known to control lipogenesis and lung maturation. Fabp5 is observed in multiple cell types of the lung, all of which possess strong lipogenic programs (TII cells, lipofibroblasts, and macrophages). The murine and human Fabp5 promoters contain multiple putative regulatory elements that could induce lung and lipid-specific Fabp5 expression. Finally, Fabp5 is down-regulated in multiple murine models of defective lung maturation, deficient surfactant production, or improper epithelial differentiation.

Ideally, understanding the function and regulation of Fabp5 during perinatal lung maturation will enable better elucidation of how lipids are trafficked in the distal lung. Treatment and prevention of RDS and other similar pathologies will always be partially ineffective until these specific pathways regulating lung lipid homeostasis are more thoroughly understood.

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Potential TFBS on hFabp5 core and upstream promoter regions Core -1.0 kb Fabp5 +1.0 kb +2.0 kb

-1.5 kb -0.5 kb +0.5 kb +1.5 kb Upstream -3.5 kb -2.5 kb Fabp5

-4.0 kb -3.0 kb -2.0 kb

E-BOX CEBP NKX2 SREBP PPAR STAT3 NFAT Supplementary Figure 1. Human Fabp5 Promoter Region . -1.5 kb upstream of the Fabp5 transcription start site to the beginning of the second exon. Transcription factor binding sites were obtained from Matinspector and each box represents a putative binding site for the factor associate with it. The E-Box/SREBP cassettes (red box) are putative lipid- specific regulatory regions. Other mentioned regulatory regions are circled in blue. Between 2.0 and 4.0 kb 5’ to the Fabp5 transcription start site are two putative regulatory elements with TFBS for multiple TFs with known lipogenic or surfactant regulatory functions in multiple systems.

Fabp5 core promoter comparison mFabp5 -1.0 kb Fabp5 +1.0 kb +2.0 kb

-1.5 kb -0.5 kb +0.5 kb +1.5 kb hFabp5 -1.0 kb Fabp5 +1.0 kb +2.0 kb

-1.5 kb -0.5 kb +0.5 kb +1.5 kb

E-BOX CEBP NKX2 SREBP PPAR STAT3 NFAT

Supplementary Figure 2. Comparison of the murine and human Fabp5 core promoter regions encompassing from 1.5 kb upstream to exon 2 downstream of the TSS. 16

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