Daniel K. Podolsky Laboratory

Lab Members:

Jose Aguirre
Kathryn Devaney, Lab Manager
Masaru Nakano
Ian Rosenberg, Ph.D.
Oren Shibolet, MD

Address:

Massachusetts General Hospital
Jackson 719
55 Fruit St.
Boston, MA 02114
Phone: 617-726-7411

Current Projects:

Molecular Basis of Intestinal Epithelial Function: Regulatory Peptides (Growth Factors/Cytokines) and Receptors

The intestinal epithelium is sustained by a highly dynamic balance of cell proliferation, differentiation and senescence. Coordination of these processes is essential to functional and anatomic integrity. The overall goal of these studies is the delineation of the role of peptide growth factors in modulating proliferation and differentiation of intestinal epithelial cells at a molecular level. A major thrust of these efforts has been the examination of the regulation of expression of transforming growth factors alpha and beta (TGF K and J) fibroblast growth factors and cytokines by intestinal epithelial cells and their role in effecting interactions between these cell populations and their extracellular matrix. These studies will serve as a paradigm to understand the role of the cell surface receptors and cell matrix components in growth modulation by these peptide growth factors.

In addition to a detailed examination of the role of previously recognized peptide growth factors, important goals are the molecular characterization of novel and biologically complementary proteins with potent growth regulating activity which are expressed by intestinal epithelial cells. In aggregate, these studies will provide insight into the spectrum of factors maintaining mucosal epithelial homeostasis and the mechanisms which play a role in the complex coordination of this network.

Role of Trefoil Peptides in the Gastrointestinal Tract

Goblet cells are abundant throughout the gastrointestinal tract and play an important role in sustaining the structural and functional integrity of the mucosal surface. Although it has been long recognized that a major component of the goblet cell secretory apparatus which produces a dense viscoelastic coat present in a continuous fashion through out the gastrointestinal tract are large, densely glycosylated mucin glycoproteins, the functional contribution of the goblet cell population and the molecular basis of the specificity of gene expression within these cells have not been defined. Recently the trefoil peptide family of structurally distinct small peptides specifically expressed in a regionalized fashion at all levels of the gastrointestinal tract have been identified as a previously unappreciated major product of goblet cell populations. Circumstantial evidence implicate the importance of these proteins in preservation of mucosal integrity and modulation of mucosal function; marked induction is consistently observed in association with injury throughout the gastrointestinal tract. These peptides are categorically distinct from other modulatory peptides in their targeted secretion onto the mucosal surface where they retain biological activity due to intrinsic resistance to intraluminal degradation. However, there is little insight into the functional properties of these peptides and the molecular basis of their specific expression in a highly cell and tissue specific manner. The main goal of the studies is the elucidation of the features of the genes encoding the trefoil factors and characterization of their protein products in order to both define the molecular basis of goblet cell specific gene expression delineate the role of these abundant peptides in sustaining mucosal integrity and function. Comprehensive study of the rat and human intestinal trefoil factors (RITF and HITF), identified and cloned in this laboratory which are present in high concentrations in goblet cells throughout the small and large intestinal epithelium is being undertaken. Major specific aims include: (I) Identification of genetic elements responsible for the highly specific expression of intestinal trefoil factor in intestinal and colonic goblet cells as well as genetic elements contributing to functional modulation of ITF expression. The latter will be facilitated through the combined application of transient transfection techniques and the study of ITF expression in established cell lines. (II) The functional characterization the biological activities of ITF obtained by direct purification and expression cloning are being defined using rat and human intestinal and colonic cell lines, as well as in vitro organ culture. These studies will be complemented by the assessment of the level of expression of ITF in mucosa from patients with inflammatory bowel disease and other disorders. Collectively, these studies should provide insights into the trefoil family of proteins and a newly recognized dimension of mucosal biology as well as fundamental insights into the molecular basis of intestinal and goblet cell function. These insights promise to provide new perspective on mechanisms of mucosal destruction, repair and function in inflammatory bowel disease and other forms of injury in the gastrointestinal tract.

Mucosal-Microbial Interaction.

LPS elicits several immediate proinflammatoy responses in peripheral blood leukocytes via a recently described pathway including CD14, Toll-like receptors (TLR), serine-threonine kinases, and NF-kappaB transcription factor. However, the functional responses of intestinal epithelial cells (IEC) to stimulation with LPS are unknown. Expression of mRNA and protein for CD14 and TLRs were assessed by RT-PCR, immunoblotting, and immunohistochemistry in mouse and human IEC lines. LPS-induced activation of signaling pathways (p42/p44 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), p38, p65, NF-kappaB) were assessed by immunoblotting and gel shifts. CD14 mRNA and protein expression were not detectable in IEC. However, human TLR2, TLR3, and TLR4 mRNA were present in IEC. TLR4 protein was expressed in all cell lines; however, TLR2 protein was absent in HT29 cells. Immunofluorescent staining of T84 cells demonstrated the cell-surface presence of the TLRs. LPS-stimulation of IEC resulted in activation (>1.5-fold) of the three members of the MAPK family. In contrast, LPS did not significantly induce activation of JNK and p38 in CMT93 cells, p38 in T84 cells and MAPK and JNK in HT29 cells. Downstream, LPS activated NF-kappaB in IEC in a time-, dose-, and serum-dependent manner. IEC express TLRs that appear to mediate LPS stimulation of specific intracellular signal transduction pathways in IEC. Thus, IEC may play a frontline role in monitoring lumenal bacteria.

Former Lab Members

  • Nicholas Barnich
  • Jiri Kalabis

Recent Publications:

  • Kalabis J, Rosenberg I, Podolsky DK. Vangl1 Protein Acts as a Downstream Effector of Intestinal Trefoil Factor (ITF)/TFF3 Signaling and Regulates Wound Healing of Intestinal Epithelium. J Biol Chem. 2006 Mar 10;281(10):6434-41.

  • Barnich N, Aguirre JE, Reinecker HC, Xavier R, Podolsky DK. Membrane recruitment of NOD2 in intestinal epithelial cells is essential for nuclear factor-{kappa}B activation in muramyl dipeptide recognition. J Cell Biol. 2005 Jul 4;170(1):21-6.

  • Barnich N, Hisamatsu T, Aguirre JE, Xavier R, Reinecker HC, Podolsky DK. GRIM-19 interacts with nucleotide oligomerization domain 2 and serves as downstream effector of anti-bacterial function in intestinal epithelial cells. J Biol Chem. 2005 May 13;280(19):19021-6.

  • Cario E, Gerken G, Podolsky DK. Toll-like receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C. Gastroenterology. 2004 Jul;127(1):224-38.

  • Otte JM, Cario E, Podolsky DK. Mechanisms of cross hyporesponsiveness to Toll-like receptor bacterial ligands in intestinal epithelial cells. Gastroenterology. 2004 Apr;126(4):1054-70.

  • Beck PL, Wong JF, Li Y, Swaminathan S, Xavier RJ, Devaney KL, Podolsky DK. Chemotherapy- and radiotherapy-induced intestinal damage is regulated by intestinal trefoil factor. Gastroenterology. 2004 Mar;126(3):796-808.

  • Beck PL, Xavier R, Wong J, Ezedi I, Mashimo H, Mizoguchi A, Mizoguchi E, Bhan AK, Podolsky DK. Paradoxical roles of different nitric oxide synthase isoforms in colonic injury. Am J Physiol Gastrointest Liver Physiol. 2004 Jan;286(1):G137-47.