Abstract

Research in my lab mainly focuses on three important areas at the interface between molecular cell biology, biotechnology, and medicine:

1. Red cell development, especially on the role of three signal transduction pathways downstream of the erythropoietin receptor in controlling terminal proliferation and differentiation of erythroid progenitor cells.
2. Hematopoietic stem cells, defining new cell surface proteins for their purification and new growth factors that support their expansion in culture.
3. Acrp30/adiponectin, a hormone we cloned that is made exclusively by fat cells and that increases fatty acid and glucose metabolism by muscle.

Recent work identified a family of adiponectin orthologs with similar functions and regulation. Newer areas concern the roles of micro RNAs in mammalian development and on the development of siRNA libraries for genome- wide analysis of signal transduction pathways.

Research Summary

Erythropoietin receptor (EpoR): Epo and the EpoR are essential for proliferation and differentiation of committed erythroid progenitors, as is the cytosolic protein-tyrosine kinase JAK-2. JAK2 binds to the EpoR cytosolic domain in the endoplasmic reticulum and facilitates its folding to promote cell surface expression. We showed that EpoRs exist on the cell surface as inactive dimers; Epo binding changes their conformation, leading to JAK2 activation. We identified several highly conserved amino acids in the EpoR cytosolic domain that are essential for Epo activation of JAK2 and now are using biophysical techniques to obtain the structure of this EpoR/ JAK2 complex and learn how JAK2 becomes activated by Epo binding.

The EpoR transmembrane domain is crucial in receptor activation; we have generated multiple mutations in this region that lead to constitutive (Epo- independent) activation of the receptor. Several of these convert residues near the exoplasmic side of this segment into cysteines and lead to the formation of a disulfide bond that links two receptors into a dimer. These receptors lead to activation of all normal EpoR signaling pathways and their structure presumably reflects that of the active dimeric receptor. We are synthesizing peptides corresponding to these dimeric active transmembrane a- helixes and will determine their structure by NMR. This should shed light on the structure of the Epo- activated receptor transmembrane domain.

JAK2 activates many signaling proteins including PI-3’ kinase, the transcription factor STAT5, and the Ras pathway. These pathways interact to prevent apoptosis of committed erythroid progenitors allowing them to undergo a predetermined program of terminal proliferation and erythroid differentiation. STAT5 directly activates transcription of the anti-apoptotic protein bclx. STAT5 -/- mice exhibit fetal anemia and increased apoptosis of erythroid progenitors caused by reduced bclx levels. Adult STAT5 -/- mice are anemic and deficient in generating high erythropoietic rates in response to stress. Thus Stat5 controls one rate-determining step regulating early erythroblast survival.

EpoR- activated antiapoptotic pathways downstream of PI-3’ kinase and ras have remained enigmatic. Last year we showed that activation of the PI-3’ kinase pathway leads to phosphorylation and inhibition of FOXO3a, a member of the Forkhead transcription factor family. FOXO3a, in turns, activates transcription of Tumor Necrosis Factor Apoptosis-Inducing Ligand (TRAIL). We showed that inhibition of TRAIL production following EpoR activation partially rescues cells from apoptosis, demonstrating the importance of this pathway in red cell formation.

Our current studies on Ras and other signaling pathways make use of a new culture system where fetal liver erythroid progenitors undergo normal terminal proliferation and differentiation that can be followed on a cell- to- cell level by FACS. As example, expression of a dominant- negative H-ras in CFU-E progenitors did not affect erythroid differentiation, indicating that the Ras pathway is not essential for erythroid development. But oncogenic H-ras blocked terminal erythroid differentiation and induced abnormal proliferation of CFU-E progenitors and early erythroblasts, a model for induction of leukemias by oncogenic Ras. This new culture system enables us to dissect the functions of the many signal transduction pathways downstream of Ras in this process. As a first step we found that three major pathways are abnormally activated by oncogenic H-Ras: Raf/ERK, PI3-kinase/Akt and RalGEF/RalA. However, only constitutive activation of the MEK/ERK pathway could recapitulate all of the effects of oncogenic H-Ras expression in blocking erythroid differentiation and inducing Epo-independent proliferation. Moreover, the effects of oncogenic H-Ras expression on primary erythroid cells were blocked by the addition of a specific inhibitor of MEK, allowing normal terminal erythroid proliferation and differentiation. Our data suggest that the MEK/ERK MAP kinase pathway is not essential for normal Epo- stimulated erythroid development, but constitutive MEK/ERK signaling leads to impaired erythroid differentiation. Interruption of constitutive MEK/ERK signaling is a potential therapeutic strategy to correct myeloid disorders.

Little is known concerning the degradation of Epo in the body – where this occurs or what may control it. We study the mechanism of Epo degradation, both in erythroid cells expressing the EpoR and in mice expressing abnormal numbers of Epo receptors in various tissues. One goal is to explain why certain commercially- important mutant Epo’s with extra carbohydrate chains have a longer biological lifetime. Another is to develop and test a mathematical model relating the production of Epo by the kidney at different levels of oxygen to the concentration of circulating Epo to the response of the bone marrow in inducing production of an appropriate number of red blood cells. To date we showed that some of the Epo bound to surface receptors is internalized by endocytosis and degraded in lysosomes. Most, however, either dissociates from the surface receptor into the medium or is internalized but resecreted. Long- lived Epo dissociates more rapidly from surface Epo receptors, and this may explain its longer half- life in vivo. To test this we will be examining the fate of Epo and its long- lived variants in mice with altered numbers of Epo receptors in both hematopoietic and non-hematopoietic cells; in this way he should discern the role of surface EpoRs in normal Epo turnover.

Hematopoietic stem cells: Hematopoietic stem cells (HSCs) are self-renewing and pluripotent; they reconstitute all blood and immune cell populations. For several years we have been studying these very rare cells in the fetal liver and bone marrow. We have identified two new cell surface markers for these cells – endoglin and normal prion protein - and are using them to develop new methods for their purification. In parallel we have identified several new growth factors for these cells and currently are optimizing conditions for robust expansion of hematopoietic stem cells in culture. We have cloned many novel secreted proteins expressed specifically by lines of stromal cells that support stem cell maintenance, including several novel cytokines. In parallel, we discovered a novel and rare population of CD3+ E15.5 fetal liver cells that support expansion of fetal liver HSCs in culture. DNA array experiments showed that IGF - 2 is specifically expressed in these fetal liver cells. Indeed, culture of purified HSCs with IGF-2 together with other cytokines led to a >10- fold increase in HSC numbers and activity. Thus fetal liver CD3+ cells are a novel population that are capable of supporting HSC expansion, and IGF – 2, produced by these cells, stimulates ex vivo expansion of both fetal liver and adult bone marrow HSCs.

Last year we identified Endoglin, an ancillary TGF-ß receptor, as a surface marker for long-term repopulating mouse bone marrow HSCs. Endoglin and several other genes differentially expressed on an enriched HSC population were identified using a novel PCR amplification protocol and microarray analysis. Recently we derived simple and highly efficient schemes for LTR-HSC purification using endoglin as a marker. In particular, almost all Endo+ Sca-1+ Rh^low (Rhodamine-123 low) cells are LTR-HSCs; this defines a simple and effective procedure for purifying a nearly homogenous stem cell population from mouse bone marrow. We are identifying the integrins and other adhesive proteins on these rare cells by a combination of proteomic, transcriptional profiling, and immunodetection assays.

Hormones controlling fatty acid and glucose metabolism. In 1995 we cloned a novel adipocyte- specific secreted protein hormone, Acrp30/ adiponectin, that is linked genetically and physiologically to development of Type II (adult- onset) diabetes and cardiovascular disease. Adiponectin potently induces fat and glucose catabolism by muscle, enhances glycogen accumulation in muscle, and inhibits gluconeogenesis in liver. We have shown that these events are linked to activation of AMP-activated protein kinase (AMPK) and the NF-kB signaling pathways. Currently we study the roles of the different adiponectin isoforms found in serum and the functions of a novel adiponectin receptor we recently cloned. We are cloning several other adiponectin receptors and studying how they activate different intracellular signaling pathways.

Recently we identified seven novel human and mouse proteins homologous in sequence and presumed structure to adiponectin and showed that several activate the AMPK signaling pathway. Like that of Acrp30/ adiponectin, expression of four of these homologs is far higher in adipose tissue than in any other tissue tested. Like that of Acrp30/ adiponectin, expression of these 4 genes is decreased in adipose tissue from obese mice and is downregulated by treatment with TNF-a and upregulated by treatment with a thiazolidinedione agonist of PPAR-g. Currently we are investigating their roles in glucose and lipid metabolism and are cloning their receptors.

New signaling pathways and new technologies. Regulated cleavage and release of the extracellular domain (ECD, "ectodomain shedding") of a multitude of transmembrane membrane precursors of secreted growth factors has been linked to the regulation of several signaling pathways. We are using a novel expression cloning strategy to identify novel signal transduction proteins regulating ectodomain shedding of growth factors on the cell surface.

Small interfering RNAs (siRNAs) potently silence expression of target genes. In principle siRNA libraries can be used to perform effective genome-scale functional genetic screens in mammalian cells, but their development has been hampered by the need to chemically synthesize thousands of oligonucleotides and to incorporate them into viral vectors. We have developed a technology to efficiently convert a double stranded cDNA library into a retroviral siRNA library in which siRNAs are produced in infected cells at high levels and that efficiently block expression of their target genes. Further development of this technology will greatly increase our ability to uncover new components in all of the signaling pathways we study.

Recent Publications

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Ghaffari S., L. J. S. Huang, J. Zhang and H. F. Lodish. Erythropoietin Receptor Signaling Processes, in "Erythropoietins and Erythropoiesis: Molecular, Cellular, Preclinical, and Clinical Biology", Graham Molineux, MaryAnn Foote, and Steven Elliott, editors, Birkhauser Publishing, (2003).

Ketteler, R., C. S. Moghraby, J. G. Hsiao, O. Sandra, H. F. Lodish, and U. Klingmüller The cytokine-inducible SH2 domain containing protein CIS negatively regulates signaling by promoting apoptosis in erythroid progenitor cells. J. Biol. Chem. 278: 2654 - 2660 (2003).

Bogan, J., N. Hendon, A. McKee, T-s Tsao, and H.F. Lodish Functional cloning of TUG as a regulator of GLUT4 glucose transporter trafficking. Nature 425: 727 – 733 (2003).

Ruan, H., H. J. Pownall, and H. F. Lodish. Troglitazone antagonizes TNF-a-induced reprogramming of adipocyte gene expression by inhibiting the transcriptional regulatory functions of NF-kB J. Biol. Chem. 278: 28181 - 28192 (2003).

Ruan, H. and H. F. Lodish. Insulin Resistance in Adipose Tissue: Direct and Indirect Effects of Tumor Necrosis Factor-a. Cytokine and Growth Factor Reviews 14: 447-455 (2003).

Gimeno, R. E., A. Ortegon. S. Patel, S. Punreddy, P. Ge, Y. Sun, H. F. Lodish, and A. Stahl Identification of a Heart-specific Fatty Acid Transport Protein J. Biol. Chem. 278: 16039 - 16044 (2003).

Zhang, J., M. Socolovsky, A. W. Gross, and H. F. Lodish. Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: functional analysis by a flow cytometry-based novel culture system. Blood 102: 3938 - 3946 (2003).

Tsao, T-s., E. Tomas, H. E. Murrey, C. Hug, D. H. Lee, N. B. Ruderman, J. E. Heuser, and H. F. Lodish. Role of Disulfide Bonds in Acrp30/Adiponectin Structure and Signaling Specificity: Different Oligomers Activate Different Signal Transduction Pathways. J. Biol. Chem. 278: 50810 - 50817 (2003).

Ghaffari, S., Z. Jagani, C. Kitidis, H. F. Lodish and R. Khosravi-Far. Cytokines and BCR-ABL Mediate Suppression of TRAIL-Induced Apoptosis through Inhibition of FOXO3a Transcription Factor. Proc. Natl. Acad. Sci. USA 100: 6523 - 6528 (2003).

Gimeno, R. E., D. J. Hirsch, S. Punreddy, Y. Sun, A. M. Ortegon, H. Wu, T. Daniels, A. Stricker-Krongrad, H. F. Lodish, and A. Stahl. Targeted Deletion of Fatty Acid Transport Protein-4 Results in Early Embryonic Lethality. J. Biol. Chem. 278: 49512 – 49516 (2003).

Ruan, H., M. Zarnowski, S. Cushman, and H. F. Lodish. Standard isolation of primary adipose cells from mouse epididymal fat pads induces inflammatory mediators and down-regulates adipocyte-genes. J. Biol. Chem. 278: 47585 - 47593 (2003).

Choong, M. L., A. Tan, B. Luo, and H. F. Lodish A novel role for proliferin-2 in the ex vivo expansion of hematopoietic stem cells FEBS Letters 550: 155 – 162 (2003).

Chen, C-Z., L. Li, M. Li, and H. F Lodish The EndoglinPositive Sca-1Positive RhodamineLow phenotype defines a near homogeneous population of long–term repopulating hematopoietic stem cells. Immunity 19: 525 - 533 (2003).

Zhang, C-C and H. F. Lodish. Insulin-like growth factor 2 expressed in a novel fetal liver cell population is a growth factor for hematopoietic stem cells Blood 103: 2513 - 2521 (2004).

Lodish, H. F., A. Berk, P. Matsudaira, C. Kaiser, M. Krieger, M. Scott, L. Zipursky, and J. E. Darnell. Molecular Cell Biology, 5th ed. Scientific American Press, N.Y. (2004).

Chen, C-Z., L. Li, H. F. Lodish, and D. P. Bartel. MicroRNAs Modulate Hematopoietic Lineage Differentiation. Science 303, 83-86 (2004).

Tsao, T-s, C. Hug, and H. F. Lodish. Adipokines: Regulators of Metabolic Integration and Energy Metabolism. Chapter 65 in Diabetes Mellitus: A Fundamental and Clinical Text. Third Edition. D. LeRoith, S. Taylor, and J. Olefsky eds. Lippincott Williams and Wilkins pp 963 - 978 (2004)

Luo, B., A. Heard, and H. F. Lodish. siRNA production by enzymatic engineering of DNA (SPEED) Proc. Natl. Acad. Sci. USA 101: 5494 - 5499 (2004).

Ruan, H. and H. F. Lodish. Role of Adipose-Tissue-Derived Hormones and Inflammatory Cytokines in Obesity-Linked Type 2 Diabetes Curr. Opin Lipidology 15:297-302 (2004).

Marszalek, J. R., C. Kitidis, A. Dararutana and H. F. Lodish Acyl CoA Synthetase 2 (ACS2) Over-expression Enhances Fatty Acid Internalization and Neurite Outgrowth J. Biol. Chem. 279: 23882 - 23891 (2004).

Kim, J., R. E. Gimeno, T. Higashimori, H-J. Kim, H. Cho, ,S. Punreddy, R. Mozell, G. Tan, A. Stricker-Krongrad, D. J. Hirsch, J. J. Fillmore, Z-X. Liu, J. Dong, G. Cline, A. Stahl, H. F. Lodish, and G. I. Shulman. Inactivation of Fatty Acid Transport Protein 1 Prevents Fat-Induced Insulin Resistance In Skeletal Muscle J. Clinical Investigation 113: 756 - 763 (2004).

Choong, M. L., B. Luo, and H. F. Lodish. Microenvironment- driven changes in the expression profile of hematopoietic cobblestone area- forming cells. Ann. Hematol. 83:160–169 (2004).

Choong, M. L., Y. P., Yonga, A. Tana, B. Luo, and H. F. Lodish. LIX: a chemokine with a role in hematopoietic stem cells maintenance. Cytokine 25: 239 – 245 (2004).

Zhang, J., and H. F. Lodish Constitutive activation of the MEK/ERK pathway mediates all effects of oncogenic H-ras expression in primary erythroid progenitors. Blood 104: 1679 – 1687 (2004).

Hug, C., J. Wang, N. Ahmad, J. Bogan, T.-S. Tsao, and H. F. Lodish. T-cadherin is a receptor for hexameric and high molecular weight forms of Acrp30/adiponectin. Proc. Natl. Acad. Sci. USA 101: 10308 - 10313 (2004).

Tong, W., and H. F. Lodish. Lnk inhibits Tpo/mpl signaling and Tpo-mediate megakaryocytopoiesis. J. Exp. Med. 200: 569 - 580 (2004).

Wong, G., J. Wang, C. Hug, T.-S. Tsao, and H. F. Lodish. A family of Acrp30/adiponectin structural and functional paralogs Proc. Natl. Acad. Sci. USA 101: 10302 - 10307 (2004).

Chen, C-Z., and H. F. Lodish. microRNAs as regulators of mammalian hematopoiesis. Semin. Immunol. in the press (2004).

Hug, C. and H. F. Lodish. The role of the adipocyte hormone Adiponectin in cardiovascular disease. Current Opinions in Pharmacology in the press (2004).

Hug, C. and H. F. Lodish. Visfatin: a new adipokine (Perspective) Science in the press (2004).

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