OASIS AND XBP-1 ACTIVITY IN OSTEOBLAST DIFFERENTIATION AND OSTEOSARCOMA By AARON BRADFORD BRISTER Submitted in partial fulfillment of the requirements For the degree of Master of Science Department of Physiology and Biophysics CASE WESTERN RESERVE UNIVERSITY January, 2008 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Aaron Bradford Brister Candidate for the Master’s degree. (signed) Corey Smith (Chair of the Committee) Clark Distelhorst Edward Greenfield George Dubyak Stephen Jones Susanne Mohr (Date) 11/20/2007 2 TABLE OF CONTENTS TABLE OF FIGURES ...................................................................................................... 5 ACKNOWLEDGEMENT ................................................................................................ 6 Abstract ............................................................................................................................... 7 1. Introduction ..................................................................................................... 8 2. Osteoblast Role in Bone Matrix Production ................................................. 9 3. The Endoplasmic Reticulum ........................................................................ 11 3.1. The Unfolded Protein Response ........................................................................ 12 3.1.1. Protein Folding and ER Chaperones .................................................... 15 3.1.2. UPR Stress Sensor Activation and OASIS Relevance ......................... 16 3.1.3. UPR Transducers: ATF6, PERK, and IRE1 ........................................ 18 3.1.4. XBP-1: A UPR Stress Signaling Molecule ............................................ 19 3.1.5. OASIS: A UPR Stress Transducer ........................................................ 21 4. The Role of OASIS and XBP-1 in Osteoblast Bone Matrix Secretion ..... 24 5. Known Characteristics of Osteosarcoma ................................................... 29 6. Hyper OASIS and XBP-1 Activity: Enabling Osteosarcoma via Elevated GRP78? .......................................................................................................... 29 6.1. The Role of GRP78 in Cancer Progression and Metastasis ............................ 31 6.2. Mechanisms of GRP78 Cell Death Inhibition .................................................. 32 6.3 Mechansims of Upregulating GRP78 ................................................................ 33 6.4. The Link between Dedifferentiated Osteoblasts, OASIS, and XBP-1 .......... 34 6.4.1. The Genetic Links between OASIS and GRP78 Overexpression ...... 35 3 6.4.2. The Link between XBP-1 and GRP78 Overexpression ....................... 36 7. Conclusions ................................................................................................................ 38 8. Future Directions ...................................................................................................... 40 References ........................................................................................................................ 53 4 TABLE OF FIGURES Figure 1 | Osteoblast differentiation signaling: from mesenchymal stem cells (MSCs) to mature osteoblasts.. .................................................................................. 46 Figure 2 | The pathways within the endoplasmic reticulum (ER) that assist protein quality control.. ............................................................................................. 47 Figure 3 | Endoplasmic reticulum (ER) stress and Unfolded Protein Response signaling.. ....................................................................................................... 48 Figure 4 | Frame switch splicing of XBP-1 by activated IRE1.. .................................. 50 Figure 5 | A model of osteoblast differentiation signaling inducing OASIS and XBP- 1....................................................................................................................... 51 Figure 6 | A hypothetical mechanism of Retinoblastoma Protein (pRB) inactivation conferring increases in OASIS and XBP-1 expression. ............................. 52 5 ACKNOWLEDGEMENT I would like to thank my parents Aubrey and Denise Brister, my brother Matthew, and my girlfriend Lauren Ebling for their love, unfailing support, and encouragement. 6 OASIS and XBP-1 Activity in Osteoblast Differentiation and Osteosarcoma Abstract By AARON BRADFORD BRISTER The Unfolded Protein Response (UPR) within the Endoplasmic Reticulum (ER) is a quality control mechanism ensuring properly folded proteins. OASIS and XBP-1 are two signal carriers of the UPR. The UPR is linked to tissue and cancer development. Elevated OASIS and XBP-1 activity are observed within developing osteoblasts. Additionally, heightened OASIS activity is present within osteosarcoma while sustained XBP-1 activity, unreported in osteosarcoma, is observed in other cancers. These signaling proteins may mediate development of mature osteoblasts and osteosarcomas. Therefore, evidence is presented with supporting mechanistic hypotheses indicating unique functions for OASIS and XBP-1 in osteoblast differentiation and osteosarcoma. These functions include, but are not limited to, enlarging the ER, buffering unfolded protein accumulation, mitigating UPR-induced cell death, and processing soluble ER proteins destined for secretion. 7 1. Introduction The intent of this document is to examine two signaling molecules associated with the Unfolded Protein Response (UPR), a quality control mechanism within a cell’s Endoplasmic Reticulum (ER). These are Old Astrocyte Specifically Induced Substance (OASIS) and X-box Binding Protein 1 (XBP-1). OASIS and XBP-1 activities will be discussed within two contexts: osteoblast differentiation and osteosarcoma. Each context will address one of the following questions. Are OASIS and XBP-1 activities directed by osteoblast differentiation programs and do their activities promote protein folding for osteoblast secretion? Could OASIS and XBP-1 hyperactivity enable osteosarcoma? The first question concerning osteoblast differentiation will be addressed in sections two through four and develop the following novel hypotheses. First, in differentiating osteoblasts, a sublethal UPR initiated by progressive increases of protein production upregulate OASIS and XBP-1 expression levels. Secondly, in maturing osteoblasts, the signaling pathways driving osteoblast differentiation also induce OASIS and XBP-1 expression so progressive increases of protein expression are matched with increases of OASIS and XBP-1 expression. Alternatively, both hypotheses may occur together. Regardless, either hypothesis prepares an osteoblast’s ER for elevated protein production and subsequent secretion, their primary functions. Further hypotheses will suggest specific functions of OASIS and XBP-1 in assisting protein processing through the ER. The second question concerning osteosarcoma will be addressed in sections five through six and develop the following hypotheses. First, in osteosarcoma, hyper OASIS and 8 XBP-1 activity maintains steady-state protein folding within the ER so an UPR once sufficient to induce cell death is now sublethal. Secondly, if differentiating osteoblasts do upregulate OASIS and XBP-1, then perhaps osteosarcomas may exploit this pathway to maintain steady-state protein folding within the ER and mitigate cell death. Additional hypotheses will examine how OASIS and XBP-1 hyperactivity reduces an UPR to a sublethal level. Also, an additional hypothetical mechanism of how OASIS is overexpressed in osteosarcoma will also be proposed. 2. Osteoblast Role in Bone Matrix Production Bone is composed of cell types derived from an embryonic germ layer. The mesoderm is an embryonic germ layer which produces Mesenchymal Stem Cells (MSCs). MSCs are self-renewing and multipotent in nature. Otherwise stated, MSCs regenerate through mitotic division or give rise to many cell types. MSCs develop into osteoblasts, bone forming cells that synthesize and secrete osteoid tissue (see figure 1). Osteoid tissue is created when proteins exuded by osteoblasts, together with minerals, form bone scaffolding. Bone proteins interact with calcium, magnesium, and phosphate ions forming hardened mineral matrix, i.e. hydroxyapatite. After an osteoblast finishes forming bone it becomes quiescent, also known as a lining cell [1, 2]. MSCs become mature osteoblasts which are “fully differentiated cells responsible for the production of the bone matrix” [3]. MSCs become osteoblasts through a complex differentiation program and many markers and signaling pathways of this program have been characterized. These markers are available for analysis and indicate the progression 9 of MSC differentiation. Once a MSC becomes a differentiating osteoblast it actively synthesizes and simultaneously secretes osseous tissue or osteoid matrix proteins which construct the skeletal system. Osseous tissue proteins include, but are not limited to, collagen type 1 IA, Alkaline Phosphatase, Osteopontin, and Osteocalcin. Important signaling molecules within differentiating osteoblasts are Runt-Related Transcription Factor 2 (RUNX2), Retinoblastoma protein (pRB), and Osterix ([4, 5] and references therein). A MSC is committed to become an osteoblast when it expresses both the intracellular signaling proteins RUNX2 and Osterix. RUNX2 (or Cbfa1) is a member of the RUNT transcription factor family. RUNT proteins have a DNA-binding