RESEARCH

Project I. Steroid-regulated exocytosis in osteoblasts: Signaling pathways in bone formation

Research in my laboratory focuses on molecular mechanisms of non-genomic effects of steroid hormones in bone cells. More specifically, we study signaling networks underlying membrane-related actions of bone anabolic steroids such as 1a,25-dihydroxyvitamin D₃ (1,25D) and estrogens in osteoblasts, the bone-forming cells. We are interested in the modulation of ion channel activities and exocytosis by these hormones in relation with bone formation. A major aim is to contribute to the understanding and treatment of some human clinical disorders of the skeleton characterized by decreased mineralization and bone mass loss, such as rickets and osteoporosis.

We study with electrophysiology 1,25D modulation of calcium (Ca²⁺) and chloride (Cl^-) currents in osteoblasts. Recently, we proposed the first physiological model for the regulation of exocytosis by 1,25D). My short-term goal is to investigate the signaling pathways involved in 1,25D modulation of ion channel and secretory activities in osteoblasts, and the molecular identity of steroid-sensitive Ca²⁺ and Cl^- channels.

 

Fig. 1. Whole-cell patch-clamp recordings

obtained from a single osteoblast. Increasing

physiological concentrations of 1,25D

promote a dose-dependent potentiation of

Cl^- currents. Control raw data show inward

L-type Ca²⁺ currents before hormone

treatment.

 

  

 

 

 

 

A                                 B                     C                                 D

  

 

 

 __ 2 mm          

 

                                                                                     __ 20 mm

Fig. 2. Series of confocal images showing 1,25D-regulated exocytosis. (A-C) were captured every 0.33 sec. The arrow shows diffusion of the fluorescent dye quinacrine into the external medium upon fusion of an individual secretory granule to the plasma membrane of a single osteoblast, shown in (D). Exocytosis occurred within the first minute after hormone treatment.

 

 

Fig. 3. Calcium signals induced by 1,25D in osteoblasts. Intracellular calcium signals were obtained with the calcium-sensitive dye Fluo-4 on a multi-plate reader.

 

Project II. Anti-apoptotic and anti-proliferative actions of vitamin D₃ in osteoblasts

Vitamin D₃ exerts relevant effects on osteoblasts, the bone-forming cells. These include protection against apoptosis and/or anti-tumor actions depending on the intensity of the hormonal stimulus. My laboratory investigates the signaling pathways leading to one effect or the other. We have characterized a rapid vitamin D₃-initiated PI3K/Akt cascade that leads to a reduction of osteoblast cell death. These anti-apoptotic actions include phosphorylation of Bad protein and FKHD transcription factors, and decreased caspase activities. These results help explain bone anabolic properties of this steroid hormone. In addition, we have discovered that a sustained treatment with hormone reduces osteosarcoma cell proliferation via a JNK/AP-1/p21 pathway. Our results reveal a potential anti-cancer effect of the hormone in bone cancer.

Fig. 4. Flow cytometry data obtained for 1,25D-anti apoptotic effects on staurosporine-induced apoptosis in osteoblasts.

 

Project III. Steroid-regulated exocytosis in glioma cells: Neuroprotective effects

We are currently studying the rapid stimulation of ion channel activities by vitamin D₃. This couples to exocytosis in NG108-15 neuroblastoma-glioma cells, and may be related to neuroprotective effects of the hormone. We are interested in characterizing the molecular signaling underlying these properties of the steroid in nervous tissues in relation with cell death and cell proliferation.

 

 

Fig. 5. Neuroblastoma-glioma cells express the vitamin D receptor (VDR). Immunofluoresce for the subcellular localization of the VDR in NG108-15 cells.

 

Purpose of our research:

The development of new therapies for treatment and prevention of bone mass loss depends on a full understanding of the molecular mechanisms by which osteoblasts produce and secrete bone, and regulation of these processes by hormones such as 1,25D and estrogen. The innovative aspect of our project resides in studying molecular mechanisms of secretion of bone materials in single, live osteoblasts. To achieve this, we utilize techniques of high spatial-temporal resolution applied to individual osteoblasts in culture such as patch-clamp electrophysiology and confocal microscopy, as well as molecular techniques applied to homogeneous populations of cells. Secretory activities are recorded in real time by means of single-cell capacitance measurements during pharmacological manipulation of signaling molecules.  Although the primary focus of our project is on basic research, the long-term objective is to identify molecular targets in the treatment of bone pathologies characterized by decreased bone mass and mineralization. This typifies skeletal diseases such as osteoporosis and osteomalacia, respectively. Osteoporosis in particular affects a large sector of the aging American population and constitutes a significant financial burden for the society.

 

SATELLITE PROJECT

Bioengineering: Growth of bone cells on carbon nanotubes

Bone structure and function depend intimately on the arrangement of cellular and non-cellular components at the nanoscale level. Bone bioengineering is aimed at creating artificial nanostructures with the capacity to increment or replace the natural tissue. The purpose of our study is to explore the use of carbon nanotubes (CNTs) as an adequate 3D-scaffold material for the growth and proliferation of bone cells and formation of functional bone tissue.  More specifically, we investigate morphological changes, cell proliferation, and integrity of the plasma membrane of osteoblasts grown on chemically functionalized CNTs.

 

 

 

 

 

 

 

 

 

 

Fig. 6. Osteoblasts growing on multi-walled CNTs developed round cell bodies and long cytoplasmic prolongations.

  

Recent press releases:

 

-          United Press International, Hi-Tech: “Nano World: Bone Cells Grow on Carbon Nanotubes”, by Charles Q. Choi, 3/24/06 (www.upi.com/Hi-Tech/view.php?StoryID=20060324-123330-6794r)

-          Small Times, NanoCom International, “Study: Bone Cells Grow on Carbon Nanotubes”, , 3/16/06 (http://www.smalltimes.com/document_display.cfm?document_id=11060)

-          Nano  Science and Technology Institute (NSTI), Cambridge, MA, “California Researchers Successfully Grow Bone Cells on Carbon Nanotubes”, by Jennifer Rocha (plus photo of the month), 3/28/06 (www.nsti.org/news/archive/0603.html)

-          Nano World News, Live Science Health Science and Technology, “Growing Bones: New Technique Proves Promising”, by Scott Fields, 3/9/06 (www.livescience.com/humanbiology/060509_growing_bones.html)

-          UCR Newsroom, “UCR Researchers Grow Bone Cells on Carbon Nanotubes”, by Ricardo Duran, 3/14/06 (www.newsroom.ucr.edu/cgi-bin/display.cgi?id=1273)

-          BioTechnolog.pl, “Kosci na naotululach”, by Marta Pieszko, Poland, 9/4/06

-          China Technology Papers On Line, “California University at Riverside: Researchers Have Developed…”, by Tan Hoi, 3/15/06 (www.cutech.edu.cn)

-          Science Daily, “Researchers Grow Bone Cells on Carbon Nanotubes” (www.sciencedaily.com)

-          NIL Technology, Kongens Lyngby, Denmark (http://news.yahoo.com/s/space/20060510/sc_space/growing bones....)

-          BioSpace, “University of California, Riverside: News from the BioSpace Beat: Researchers Grow Bone on Carbon Nanotubes”, UC Berkeley Press (www.biospace.com/news_company.aspx?companyID=287704)

-          National Center for Nanomaterials Technology, Korea, “University of California-Riverside: Carbon Nanotube Bone Cell Growth Method Development: In Bone Defect Treatment, Bright Prospect”, 4/10/06 (www.nano.or.kr)

-          The Neurophylosopher: “Bone Cells Grow on Carbon Nanotubes” (http://neurophilosophy.worldpress.com/tag/nanotechnology)

-          Oak Ridge National Laboratory in the News: “Bone Cells Grow on Carbon Nanotubes”, 3/17/06 (www.ornl.gov/info/library/ornlnews/archives031706.shtml)

-          Intelligent Microsystem Center, Korea: “UCR Researchers Grow Bone Cells on Carbon Nanotubes”, 4/2/06 (www.microsystem.re.kr)

-          The Big Think Tank: “Bone Cells Grown on Carbon Nanotubes”, 4/2/06 (http://thebigthinktank.blogspot.com/2006/03/bone-cells-grown-on-carbon-nanotubes.html)

 

Current funding: National Institutes of Health, DK07115

 

Current open positions

Postdoctoral Researcher. Full-time research position to work on ion channel modulation by steroid hormones. The candidate will preferably have training in patch-clamp electrophysiology, cell culture, and basic molecular biology techniques. Initial contract will be for a year, starting in Spring 2007, with the possibility of renewal. PhD degree is required. Strong communication skills are required for this position. Creativity, self-motivation, and capacity to work independently are a must. Willingness to train graduate students is also expected. Please email resume and the names and contact information of three references to laura.zanello@ucr.edu. Mention this ad in your cover letter.

Graduate students. Strong graduate students seeking a PhD degree in Biochemistry, Cell and Molecular Biology, Biomedical Sciences, or Neuroscience at the University of California-Riverside are welcome to join my laboratory. The PhD research project will develop aspects of any of the projects presented in this website. Preferably, I am currently looking for a PhD student with special interest in ion channels, who is willing to train in patch-clamp electrophysiology. Pre-requisites to apply to my lab are a GPA of 3.5 or higher, and high performance in the GRE examination. If you are a highly motivated and ambitious student, this is a lab where to work. I expect full commitment to science and the life style of a researcher. Excellent writing skills will definitively open extra-doors to this graduate student.

Lab Philosophy 

My laboratory is built upon the following pillars:

-          Scientific excellence

-          Academic integrity

-          Honesty, accuracy, efficiency, objectivity

-          Self-motivation

-          Passion for science

 

SELECTED PUBLICATIONS

Zanello, L.P., and Norman, A.W. (1997) Stimulation by 1a,25(OH)2 vitamin D3 of whole-cell chloride currents in osteoblastic ROS 17/2.8 cells: A structure-function study. Journal of Biological Chemistry 272: 22617-22622

Norman, A.W., Song, X.D., Zanello, L.P., Bula, C., and Okamura, W.H. (1999) Rapid and genomic biological responses are mediated by different shapes of the agonist steroid hormone, 1a,25(OH)₂ vitamin D₃. Steroids 64: 120-128

Norman, A.W., Bishop, J.E., Bula, C.M., Olivera, C.J., Mizwicki, M.T., Zanello, L.P., Ishida, H., and Okamura, W.H. (2002) Molecular tools for study of genomic and rapid signal transduction responses initiated by 1a,25(OH)₂ vitamin D₃. Steroids 67: 457-466

Zanello, L.P., and Norman, A.W.  (2003) Molecular mechanisms of rapid 1a,25(OH)₂-vitamin D₃ multiple modulation of ion channel activities in osteoblasts. Bone 33: 71-79

Zanello, L.P., and Norman, A.W.  (2004) Rapid modulation of osteoblast ion channel responses by 1a,25(OH)₂-vitamin D₃ requires the presence of the functional vitamin D receptor. Proceedings of the National Academy of Sciences 101: 1589-1594

Zanello, L.P., and Norman, A.W.  (2004) Electrical responses to 1a,25(OH)₂-vitamin D₃ and their physiological significance in osteoblasts. Steroids 69: 561-565

Zanello, L.P., and Norman, A.W.  (2006) 1a,25(OH)₂-vitamin D₃ actions on ion channels in osteoblasts, Steroids, 71: 291-297

Zanello, L.P., Zhao, B., and Haddon, R.C. (2006) Bone cell proliferation on carbon nanotubes, Nano Letters, 6: 562-567

Zanello, L.P. (2006) Electrical properties of osteoblasts cultured on carbon nanotubes, IEE Micro and Nano Letters, 1: 19-22

Zanello, L.P. (2006) Non-genomic mechanisms of vitamin D-regulated bone formation in osteoblasts, Clinical Cases in Mineral and Bone Metabolism, 3: 50-57

Wu, W., Zhang, X., and Zanello, L.P. (2006) 1a,25(OH)₂-vitamin D₃ upregulates p21waf1 and inhibits human osteosarcoma cell proliferation via activation of MAPK pathways, Cancer Letters, in press

Zhang, X., Biswas, P., Owraghi, M., and Zanello, L.P. (2007) 1a,25(OH)₂-vitamin D₃ membrane-initiated calcium signaling modulates exocytosis and cell survival, Journal of Steroid Biochemistry and Molecular Biology, 2007 Jan 5; [Epub ahead of print]

Books (translations)

1.             Zanello L.P. and A.H. Rolan. Biology: Concepts and Relations, by Campbell, N.A., L.G. Mitchell, Reece, J.B., 3^rd edition, 2000, Pearson Educacion Latinoamerica, Mexico, translated into Spanish from original English version, 860 pages.

 

2.             Zanello, L.P. Glossary for Biology, by Campbell, N.A., Reece, J.B., 7^th edition, 2005, Benjamin Cummings Publishers, CA, translated into Spanish from original English version, 54 pages.

 Lab members: