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(*3t,U~§ 1W1F~jl~,E,) (The Journal of Japanese College of Angiology)

Vol. 4, 179-183, 2008.

Department of Cardiovascular Medicine,

Graduate School of Medicine,

The University of Tokyo, Professor Ryozo Nag

a

i

Abstract

In response to physical, metabolic and immune stress, several malformations are revealed in the cardiovascul,ar system, including hypertrophy, hyperplasia, and fibrosis. We previously reported that the transcription factor, KLF5, an activator for vascular smooth muscle, plays a pivotal role in cardiovascular remodeling, organ fibrosis, adiopocyte differentiation and lipid metabolism in skeletal muscles. Thus, we believe that agonists of retinoic acid receptor (RAR) binding to KLF5 or agonists of PPAR (5 modifying KLF5 with small ubiquitin-like modification (SUMOylation) can be used as new therapeutic target for cardiovascular diseases and metabolic syndromes.

Introduction

In order to maintain its homeostasis, the living body is continually required loJace pbalf!ic?3 s and

metabolic stress. Long-term stress may trigger the signal transduction in cells to transactivate gene transcription and further result in cell hypertrophy and proliferation. These may even cause higher-level changes in the organ structures, such as atherosclerosis, vascular restenosis, and cardiac hypertrophy, in cardiovascular organs. Obesity that is caused by excessive intake of calories may also, in some way, be interpreted as a response to the stress from adipocytes. One of the basic clinical diagnostic criteria for metabolic syndrome is the malfunction and structure changes in higher-level cardiovascular and adipose tissues. The existence of a common molecular mechanism between the cardiovascular and adipose tissues may not only be interpreted as the stress responsive mechanisms in cells, but also as an important issue in the prevention and treatment of cardiovascular diseases.

23

The Role of the Transcription Fac 0

Remodeling

In response to the process of differentiation or pathogenic pressure, a great diversity in the gene level has been observed in skeletal and cardiac muscles. In particular, the change of myosine isoforms is used as a molecular marker for muscle development and muscle diseases, and contributes differentially to contractile activity in muscles. Similarly, the myosin polymorphism in skeletal and cardiac muscles is also observed in smooth muscles and used as molecular markers for angiogenesis or diseases in vessels. Among these markers, two smooth myosin isoforms, SM1 and SM2, have been used as the best markers to identify smooth muscles due to their high specificity. These markers are also confirmed as the stress proteins induced by the embryonic form of smooth muscle myosin heavy chain (SMemb) under the stress of proliferation and oxidation responses. Furthermore, SMemb not only is shown to proliferate the smooth muscle cells in atherosclerosis or vascular restenosis, but also is induced in the process of organ remodeling in cardiac hypertrophy, glomerulonephritis or interstitial nephritis and interstitial pneumonia.

Based on previous studies, we first identified Kr i.i ppel-like factor 5 (KLF5, also referred to as BTEB2, and IKLF) as a transcription factor that regulates expression of the SMemb gene. Kr i.i ppel is an important transcription factor for segmentation in Drosophila, and KLF is its homologue gene in mammals.

The expression of the growth factor, including platelet-derived growth factors (PDGF)-AlB, activates KLF5 to induce the transcription factors to activate some gene clusters related to cardiovascular diseases, such as Egr-1 , plasminogen activator inhibitor-1 (PAI-1), etc. Moreover, KLF5 binds to the NFkB subunit p50 to enhance the activation of PDGF. Furthermore, the anti-apoptotic actions of KLF5 are mainly caused by a significant acetylation process of lysine residues in the DNA binding domain. It also has been shown that the formation of transcription factor complex by RAR, PAR and C/EBP modulate the KLF5 transcription activities. The importance of the KLF family is its novel molecular structure in response to stress and differentiation in varied tissues. For example, KLF2, 4, and 6 play an important role in the maintenance of cardiovascular function and atherosclerosis. KLF2 and 5 are very important in cell differentiation and cell­ mediated immunity. KLF5 and 15 are related to the adipocyte differentiation and, further, cause obesity and fatty liver. The KLF family can either act as a positive regulator to promote gene transcription or as a repressor to downregulate gene expression. Therefore, the same factors may act differently in a tissue­ specific manner, in either positive or negative regulation. However, the details of its mechanism remain to be elucidated.

The significant role of KLF5 in the pathogenesis of cardiovascular diseases has been confirmed by gene knockout experiments. In the KLF5 ' knockout mice, the deletion of KLF5 gene in both alleles is lethal in the early stage of embryogenesis, which makes it impossible to dissect. Although the details of the mechanisms causing the death of homozygous mutant mice remains unclear, these indicates the essential role of KLF5 in the maintenance of ES cells. Thus, it has been hypothesized that the early death of embryos may be caused by the defects occurring in early embryogenesis.

On the other hand, the heterozygous KLF5-knockout mice (KLF5+/-) were apparently normal, but showed diminished levels of arterial wall thickening , cardiac hypertrophy, and interstitial fibrosis in response to angiotensin II. Thus, KLF5 seems to be a key transcription factor in mediating external stress and cardiovascular remodeling .

In addition, KLF5 also has an angiogenesis factor. In fact, the tumor cells implanted into the KLF5 heterozygous mutant were markedly attenuated. Therefore, we propose that the angiogenesis in the normal cells is critical during the malignant hyperplasia. Thus, KLF5 is thought to be a factor to activate mesenchymal stem cells.

Roles of KLF5 in Adipocyte Differentiation and Metabolic

Syndrome

KLF5 is not only related to the initiation of cardiovascular development in response to stress, but also crucially involves the differentiation of adipocytes. It has been shown that when the full length of KLF5 cDNA transfected into 3T3L 1 cell line, that induced preadipocytes to differentiate into adipocytes. In addition, under the condition for the adipocyte differentiation, the introduction of KLF5 siRNA in 3T3L 1 cells successfully blocks the adipocyte differentiation. In the network that regulates adipocyte differentiation, KLF5 acts downstream of C/EBP but upstream of PPAR2.

It has been observed that the KLF5 knockout mutant mice were resistant to high-calorie diet-induced obesity and metabolic load. In other words, despite consuming more than twice the food of wild-type mice, the body weight increase is limited (-10%) as compared to the wild-type littermates. Only mild hyper­ glucose surges and insulin tolerance was observed. Compared to the metabolic index, the heterozygous mutants have similar carbohydrate intake to the wild-type, but the energy expenditure is highly enhanced in skeletal muscle. That means that KLF5 inhibits heat consumption in the skeletal muscles. In fact, it has been shown that several genes are involved in the lipid oxidation and energy uncoupling, including acyl-CoA oxidase in fatty acid (3 -oxidation, CPT1 b in mitochondria, and uncoupling protein 2 and 3 (UCP2, 3) in energy production. These genes Significantly upregulate in the skeletal muscle of heterozygous mutant mice. In the case of myocytes, KLF5 significantly inhibits the C/EBP-mediated transacti on-nfthf~f"!"'ntII!nfI~

In recent years, the switch mechanism by which transcription factors repress or activate genes in different tissues is becoming the focus of attention with the studies of SUMO modification in transcription factors. This is because it may be one of the mechanisms that convert transcription factors to transcription repressors in a tissue-specific manner. In fact, the SUMOylation of Lys162 and Lys209 toggle the interaction of KLF5 binding to the co-repressor NcoR or SMRT. The expression of KLF5 in skeletal muscle will be induced by high-fat food; thus, we believe that in a state of excessive intake of calories, obesity, fatty liver and hyperlipidemia will be induced; and these metabolic abnormalities may even further induce the expression of KLF5 in the cardiovascular system, causing cardiovascular diseases.

On the basis of the finding mentioned above, KLF5 can be applied in drug development. In particular, KLF5 is not acting alone to activate those pathological-related genes, but is forming a transcriptional complex with other nuclear receptors and molecules. Thus, a number of compounds were tested for their ability to modulate KLF5's trans-activation ability to use PDGF-A promoter as a molecular marker; two compounds- AmBO (Tamibarotene), a synthetic RAR agonist, and LE135, a synthetic RAR antagonist ­ were revealed in the screening. In fact, systematic administration of AmBO led to inhibited neointimal formation in injured arteries, reduced atherosclerotic lesions and foam cell accumulation in the aortas of apolipoprotein E (apoE)-deficient mice, suppressed angiotensin II-induced cardiac hypertrophy and fibrosis, and repressed formation of high-calorie diet-induced fatty Liver. On the other hand, LE135 has been shown to enhance development in angiogenesis.

In addition, the chemical compounds inhibit KLF5 activity in skeletal muscle, may have the potential to be the treatment for metabolic syndrome and arteriosclerosis. GW501516 was first developed as a drug to lower LDL and raise HDL levels, which acts as a PPAR agonist. Animals that orally administered GW501516 with high-calorie diet showed to ameliorate leading to obesity. We noticed a striking similarity between the metabolic phenotypes of KLF5 heterozygous mutant mice with the GW501516-treated mice, which has been shown to induce the expression of acyl-CoA oxidase, CPT1 b and uncoupling protein 2 (UCP2) in skeletal muscle. Upon review of its mechanism in skeletal muscle cells, it was found that KLF5 formed transcriptional regulatory complexes with fatty acid metabolism-related genes. However, in the presence of PPAR agonist GW501516, KLF5 is deSUMOlyated, followed by dissociation of the co-repressors NcoR and SMRT, and the expression of fatty acid metabolism-related genes increased.

Conclusion

Our research originated from a clear elucidation of the myosin polymorphism in the vascular smooth muscle; induction of cardiovascular diseases by SMemb; identification of KLF5, a transactivator of SMemb; explanation of the significant pathophysiological role of KLF5 in cardiovascular diseases and further initiation for drug development. After a series of studies, based on the knowledge of diseases other than cardiovascular disease and metabolic syndrome, we found that KLF5 responded to external stress, thus, the activation of KLF5 in skeletal increases the energy consumption and further lowers the incidence of metabolic syndrome. We also found that KLF is closely related to the obesity caused by adipocyte differentiation in adipose tissues, and the formation of cardiac hypertrophy, arteriosclerosis, and restenosis cardiovascular tissues.

Our study aims to provide an important point of view on this aspect of gene transcription in order to understand the molecular mechanisms of metabolic disorders and cardiovascular remodeling that contributes to metabolic syndrome and cardiovascular diseases.

Original paper from

The Journal of Japanese College of Angiology, Vol. 4, 179-183, 2008.