Douglas S. Darling, Ph.D.
Professor of Periodontics, Endodontics, and Dental Hygiene
Department of Oral Health and Rehabilitation,
Birth Defects Center,
Center for Genetics and Molecular Medicine, and
Center for Environmental Genetics and Integrative Biology
- Defining the molecular basis for trafficking of secreted proteins in salivary glands.
- Mathematical modeling of parotid gland terminal differentiation.
- Regulation of development by ZEB1.
- Dr. Venkatesh Srirangapatnam; Postdoc
- Anne Carenbauer; Technician Senior
- Melissa Metzler; PhD Student (Biochemistry)
- Diana Blau; Masters in Oral Biology Student
- Sarah Perez; Masters in Oral Biology Student
- Ian Pyle; Masters in Oral Biology Student
We are studying the molecular basis for trafficking of secreted proteins within the cell during regulated secretion. Secretory proteins synthesized in the major salivary glands, including the parotid glands, are primarily stored in dense-core secretory granules and released in response to external stimuli (regulated or stimulated secretion). Parotid salivary proteins are secreted apically, primarily by the regulated secretory pathway. Proteins that are not targeted or retained in secretory granules are secreted constitutively (including basolateral secretion). Trafficking of cargo proteins must direct them into the correct pathway. Understanding how sorting occurs in salivary glands would contribute to the correct targeting of therapeutic transgenes. To study the molecular basis for trafficking, we use a proteomics approach to define proteins in the secretory granule membrane, and several protein-interaction approaches. We use a novel model for sorting of salivary proteins in which Parotid Secretory Protein (PSP) binds the membrane at phosphatidylinositol bisphosphate.
Loss of salivary gland function is clinically important. Understanding the differentiation of salivary cells is a necessary step to enable the restoration of diseased or destroyed parotid salivary tissue. Computer modeling of biological systems is a potentially powerful approach to discriminate key regulatory steps. We are using gene expression arrays of differentiating salivary glands to identify networks that cause terminal differentiation. The gene networks are being incorporated into differential equation models of development. These computer models are expected to have the capacity to test the relative importance of specific pathways within the network, allowing further validation by laboratory work.
Separately, our laboratory is interested in the regulation of gene transcription in eucaryotic cells. We are investigating the molecular mechanism of action of the zfh family of transcription factors. We had isolated cDNA and genomic clones of the Zinc Finger Homeodomain Enhancer-binding Protein (Zfhep, Zfhx1a, ZEB1) gene. ZEB1 is essential for life as well as T-cell development, craniofacial development, and skeletal patterning. ZEB1 is a SMAD-binding protein, and therefore is part of the TGFbeta family signaling mechanism. Current projects investigate the role of ZEB and related genes in early development, and the molecular interactions that underlie those roles. We focus on development of the eye, which has specific defects in ZEB1-mutant mice that mimic birth defects in some human babies. In addition, we are studying the molecular basis for cleft palate in ZEB1 knock-out mice.
- Jin JZ, Li Q, Higashi Y, Darling DS, and Ding J (2008) Analysis of Zfhx1a mutant mice reveals palatal shelf contact-independent medial edge epithelial differentiation during palate fusion. Cell and Tissue Research, 333:29-38. doi: 10.1007/s00441-008-0612-x
- M Singh, NS Spoelstra, A Jean, E Howe, KC Torkko, HR Clark, DS Darling, KR Shroyer, KB Horwitz, RR Broaddus, and JK Richer (2008) ZEB1 expression in type I vs type II endometrial cancers: a marker of aggressive disease. Mod Pathol. 21(7): 912-23. PMID 18487993.
- Yongqing Liu, Xiaoyan Peng, Jinlian Tan, Douglas S. Darling, Henry J. Kaplan, and Douglas C. Dean (2008) Zeb1 Mutant Mice as a Model of Posterior Corneal Dystrophy. Invest. Ophthalmol. Vis. Sci., 49: 1843-1849. DOI: 10.1167/iovs.07-0789.
- Liu, Y., El-Naggar, S., Darling, D.S., Higashi, Y., and Dean, D.C. (2008) Zeb1 links epithelial-mesenchymal transition and cellular senescence. Development 135, 579-588. PMID: 18192284.
- Kowase T., Walsh H.E., Darling D.S., and Shupnik M.A. (2007) Estrogen Enhances GnRH-Stimulated Transcription of the LH Subunit Promoters via Altered Expression of Stimulatory and Suppressive Transcription Factors. Endocrinology, 148: 6083 – 6091. PMID:17823254.
- Liu, Y., Costantino, M.E., Durango-Montoya D., Darling, D.S., and Dean, D.C. (2007): The zinc finger transcription factor ZFHX1A is linked to cell proliferation through the Rb/E2F pathway. Biochem. J. 408(1): 79-85. PMID: 17655524.
- Kitchens DH, Binkley CJ, Wallace DL, Darling DS, (2007) Helicobacter pylori Infection in Intellectually and Developmentally Disabled Persons: A Review. Special Care in Dentistry. 27 (4):127-33, PMID:17972442.
- Manavella PA, Roqueiro G, Darling DS, and Cabanillas AM. (2007) The ZFHX1A gene is differentially autoregulated by its isoforms. Biochem. Biophys. Res. Commun. 360(3): 621-626. PMID:17610840.
- S.G. Venkatesh, J. Tan, S.U. Gorr and D.S. Darling (2007) Isoproterenol Increases Sorting Of Parotid Gland Cargo Proteins To The Basolateral Pathway. Am J Physiol Cell Physiol. 293: C558 - C565. PMID: 17537806.
- Imbert Y., Darling DS, Jumblatt MM, Foulks GN, Couzin EG, Steele PS, and Young, WW Jr. (2006) MUC1 splice variants in human ocular surface tissues: possible differences between dry eye patients and normal controls. Exp Eye Res, Sep 2006; 83(3): 493-501. PMID: 16631167.
- Spoelstra N, Manning NG, Higashi Y, Darling DS, Singh M, Shroyer K, Broaddus RR, Horwitz KB, and Richer JK (2006) The Transcription Factor ZEB1 is Aberrantly Expressed in Aggressive Uterine Cancers. Cancer Res., 66: 3893 - 3902. PMID: 16585218.
- Gorr, SU, Venkatesh, SG, Darling, DS (2005) Parotid Secretory Granules: Cross-Roads of Secretory Pathways and Protein Storage. J. Dental Research 84(6):500-509. PMID:15914585.
- Darling D.S., Stearman R.P., Qi Y, Qiu M., and Feller J.P. (2003) Expression of Zfhep/δEF1 protein in palate, neural progenitors, and differentiated neurons. Gene Expression Patterns 3: 709-717. PMID: 14643678
- Smith, GE and Darling, DS (2003) Combination of a Zinc Finger and Homeodomain required for protein-interaction. Mol. Biol. Reports. 30 (4):199-206. PMID:14672405.
- Costantino, M.E., Smith, G.E. and Darling, D.S. (2002) Cell-Specific Phosphorylation of Zfhep Transcription Factor. Biochem. Biophys. Res. Com. 296: 368- 373. PMID: 12163027
- Yen, G., Croci, A., Dowling, A., Zhang, S., Zoeller, R.T., Darling, D.S. (2001) Developmental and functional evidence of a role for Zfhep in neural cell development. Molecular Brain Research. 96: 59-67. PMID: 11731009.
- Cabanillas A.M., Smith G.E., Darling D.S. (2001) T3-Activation of the Rat Growth Hormone Gene is Inhibited by a Zinc Finger/Homeodomain Protein. Mol Cell Endocrinol. 181:131-137. PMID: 11476947