In the past several years research in my laboratory has focused on understanding how cells and organisms respond to stresses in the environment. Specifically, we have concentrated on the signaling pathways involved in transcriptional regulation of heat shock proteins (Hsps, also known as stress proteins or molecular chaperones) during the stress response in mammalian cells. More recently we have expanded our work to include research into animal models with targeted disruption of heat shock transcription factors (Hsfs), heat shock proteins (Hsps), and heat shock factor associated proteins in order to understand the function of these proteins in vivo. Heat shock proteins are critically involved in protein folding and higher order assembly of multimeric protein complexes, and many disease states are associated with abnormalities in protein folding, including Alzheimer's disease, Huntington's disease, Amylotrophic Lateral Sclerosis, Cystic Fibrosis, and Spongiform Encephalopathies. Furthermore, high levels of expression of molecular chaperones have been detected in many human cancers, the significance of which remains unclear.
Overall goal of our analyses using animal models. The function of heat shock proteins and other molecular chaperones in the mammalian system is under intense investigation because of their expanding roles in aging and neurodegenerative diseases as well as cancer. However, the use of animal models deficient in heat shock proteins remains in its infancy. In collaboration with Dr. Demetrius Moskophidis in the Molecular Chaperone Biology/Radiobiology Program, we have been generating animal models with targeted disruption of heat shock factors and heat shock proteins using conventional and conditional knockout strategies. Our analyses of these animal models indicate fascinating and complex roles played by heat shock factors and heat shock proteins in the differentiation and development of essential cell compartments of various tissues (2-4 cell embryos, spermatogonia, T cells, astrocytes, lens fiber cells). Utilizing the animal models we have generated and animal models currently being produced, we hope to unravel the complex function of heat shock proteins as pertains to human diseases in the next few years. This knowledge promises to contribute to novel and better molecular-based therapies to treat a wide spectrum of human illness.
We have identified MAP kinase signaling pathways in the regulation of transcriptional activity of heat shock transcription factors Hsf1 and Hsf4 in mammalian cells. Hsf1 phosphorylation by the MAP kinases ERK and JNK leads to repression of Hsf1 transcriptional activity (Mivechi and Giaccia, Cancer Res. 55:5512, 1995; He, et. al. MCB 18:6624, 1998; Dai, et. al. JBC 275:18210, 2000). Using a yeast two-hybrid screening system to identify signaling pathways that regulate Hsf1 and Hsf4 activities, we have identified Ral binding protein 1 (RalBP1) as an Hsf1 interacting protein, and have shown that RalBP1 regulates Hsf1 transcriptional activity by sequestering Hsf1 in the cytoplasm. The RalBP1 interaction with Hsf1 brings Hsf1 regulation into the Ral (a Ras family member) signaling pathway. We anticipate that signaling pathways that activate the Ral signaling pathway also lead to activation of Hsf1 and expression of Hsps (Hu and Mivechi, JBC 278:17299, 2003).
To further understand the signaling pathways modulating the activity of Hsfs and to extend this to animal models, we are determining the mechanism by which activation of the Ral signaling pathway contributes to Hsf1 activity, and whether phosphorylation of Hsf1 is required for Hsf1 release from the RalBP1-Hsf1 complex. Hsf1 is phosphorylated by ERK and glycogen synthase kinase (GSK) on serines 307 and 303, respectively. In order to understand the significance of Hsf1 phosphorylation in vivo, we have generated a knock-in mouse line where the Hsf1-specific phosphorylated serine residues have been mutated to alanines. Cells derived from this mouse line will be used to examine Hsf1 activity and Hsp gene expression. Using this mouse model, we will also establish the impact of phosphorylation of Hsf1 by MAP kinases in vivo.
To understand the functions of Hsfs in vivo, we have generated hsf1, hsf2, and hsf4 knockout mice (Zhang, et.al. JCB 86:376, 2002; Wang, et.al. Genesis 36:48, 2003; Min, et.al. Genesis, in 40:205-217, 2004). Disruption of hsf1 illuminates its importance for regulation of apoptosis, control of thermotolerance, and female fertility. In contrast, the hsf2 knockout mouse exhibits defects in development of the central nervous system and reduced spermatogenesis. We have further determined that deletion of both hsf1 and hsf2 has synergistic effects, resulting in abnormalities more severe than those manifested in individual hsf-deficient mice. These abnormalities include complete disruption of spermatogenesis leading to male sterility (Wang, et.al. Genesis 38:66, 2004). Therefore, the hsf1hsf2 doubly deficient mice exhibit both male and female sterility. Our most recent studies reveal that disruption of the hsf4 gene in mice results in the development of early onset cataract with 100% penetrance. Lens fiber cell differentiation in hsf4-deficient mice appears to be severely affected. Microarray and proteomics analyses indicate that the major target gene controlled by hsf4 in the lens is the small heat shock protein 25 kDa (hsp25) (Min, et.al. Genesis, 2004).
To further clarify the activities of Hsfs using in vivo models, we seek to identify the molecular mechanisms underlying male infertility of hsf1 and hsf2 doubly deficient mice during spermatogenesis. Our data show that the division and differentiation of male germ cells may be affected. Therefore, we will investigate hsf1, hsf2, and hsp expression and downstream target genes during cell division (mitosis and meiosis) and differentiation of spermatogonia during maturation of male mice. To examine the impact of hsf1 deficiency in spontaneous tumor development, we have crossed hsf1-deficient mice with several animal models that develop spontaneous tumors. The time required for tumor development, types of tumors that develop, and the expression of inducible Hsps and apoptotic response of cells to DNA damaging and protein damaging agents will be determined and compared. To elucidate the role of Hsf4 and its downstream target 25 kDa heat shock protein (Hsp25) in lens fiber cell differentiation, we will analyze the role of small Hsps (such as Hsp25 and crystallins) in lens fiber cell differentiation.
Current Program and Future Plans
Heat Shock Factor Binding Protein 1 (Hsbp1) is a 76-amino-acid polypeptide that interacts with Hsf1 and inhibits Hsf1 transcriptional activity during the stress response. To understand the role of Hsbp1 and Hsf-associated factors (Brahma-related gene 1, BRG1), we are analyzing the role of these proteins in activity of hsf and hsp gene expression using biochemical analyses in vitro and in animal models.
Recent studies of mouse models deficient in heat shock factors and their associated regulatory factors-Hsf1binding protein, Hsbp1, or the chromatin remodeling factor, BRG1�indicate that these proteins play essential roles during embryonic development and tumorigenesis. To address specific issues regarding the regulation and function of these genes during development, we have established the use of zebrafish as a model organism because of their accelerated embryonic development in comparison to mice. Our parallel analyses to understand the expression of Hsfs and their associated factors indicate that the mRNAs for these genes are expressed throughout early embryogenesis in zebrafish. Knockdown of hsf1 expression at the single cell stage of embryonic development using morpholino oligonucleotides leads to a severe apoptotic response in zebrafish embryos exposed to mild stress conditions (Wang, et.al., Genesis 2001). Our recent data demonstrate that when we knock down the expression of Hsbp1 or BRG1 there are severe embryonic abnormalities and associated neurodevelopmental defects, including malformation of body axis and, in some cases, defects in proper head formation, prior to eventual death. Interestingly, knockdown of hsbp1 or BRG1 results in a similar pattern of developmental defects, leading us to believe that Hsbp1 and BRG1 in zebrafish could affect overlapping signaling pathways (Eroglu, et.al., unpublished data). In addition to its role in regulating Hsf1 transcriptional activity, BRG1 plays a crucial role in regulating gene expression downstream of the Wnt signaling pathway and TCF (Ternary Complex Factor) transcription factors. Many genes in the Wnt signaling pathway upstream of TCFs have been identified, and when they are overexpressed some appear to cause axis duplication and formation of two heads. All of these genes have oncogenic potential in humans. The majority of genes located downstream of TCF in the Wnt pathway have not yet been identified. Since these genes are critical during embryonic development and some are oncogenic, their identification and characterization are required for possible future therapeutic interventions.
The future plans for projects involving zebrafish as a model organism are threefold. We intend to 1) identify the genes controlled by Hsfs and their associated factors. 2) identify proteins interacting with these factors and 3) reveal the function of these proteins during zebrafish development. The goal of this research is to understand the contribution of these proteins and their associated factors in the developing organism and during tumorigenesis.