Leonidas Tsiokas - US grants

Cadherins are membrane proteins mediating Ca-dependent homophilic cell adhesion and have been categorized into two major groups based on structural similarities. The prototype of class I cadherins, E- cadherin is expressed in epithelial cells. Its downregulation correlates with tumor metastasis and invasiveness. Class II cadherins comprise a list of almost ten very closely related molecules which lack specific sequences determining homophilic aggregation. High level expression has been documented not only in normal epithelial cells but also in loose tissues, migrating cells and neoplasms. Thus the functions of class H cadherins do not completely overlap with functions assigned to class I cadherins. The proposed study aims to identify the homophilic interface of a class II, fetal kidney-specific cadherin, K-cadherin (KCad), also known as cadherin -6 (cad6), by mutational analysis based on the crystal structure of E- and N- cadherin. A panel of mutants will be scored for their ability to abrogate homophilic interactions in the Drosophila S2 cell line. These data can be extrapolated to other members of the class II subfamily and may provide the molecular basis for the pharmacologic intervention of cadherin function. The ability of secreted forms of these mutants to inhibit kidney development will also be evaluated and these findings will provide necessary information to understand molecular mechanisms underlying the development of other organs which develop through mesenchymal- to-epithelial transitions. Such inductive interactions occur during salivary glands, lungs, hair and tooth development. Moreover, insights into potential role for cell adhesion in aberrant growth will be obtained, since KCad has been documented to be upregulated in various neoplasms, mainly kidney carcinoma.

DESCRIPTION (provided by applicant): The function(s) of the genes responsible for the vast majority of cases of autosomal dominant polycystic kidney disease (ADPKD) are unknown. Based on sequence analysis, the gene product of the polycystic kidney disease gene 1 (PKD1) has been proposed to encode a protein with a role in cell-cell and/or cell - extracellular matrix interactions while PKD2 is thought to function as a cation channel. We have shown that PKD2 physically associated with PKD1 and only in the presence of PKD1, PKD2 was able to form a Ca++ permeable cation channel. In addition to PKD 1, PKD2 was also able to associate with the transient receptor potential channel 1 (TRPC1). We now show that TRPC1 has a widespread distribution in epithelial structures, primarily the ductal cells of the kidney and liver. TRPC 1 was shown to enhance Ca++ entry in response to store depletion (or capacitative Ca++ entry, CCE). CCE is the major route by which non-excitable cells regulate their intracellular Ca++ concentration. Among the many cellular functions regulated by CCE, regulation of cAMP accumulation is a well-characterized and specific physiological target of CCE. Notably, the involvement of cAMP in cyst formation in kidney and liver epithelial cells has been well established. We propose that PKD1, PKD2 and TRPC1 assemble to a functional complex to enhance CCE. Naturally occurring mutations in PKD2 may result in the disruption of this complex and thereby in alterations in CCE. We will test our model by showing the existence of an endogenous complex and identifying protein-protein interactions responsible for complex assembly. Next, we will measure CCE in cells transfected with PKD1, PKD2 and TRPC1. We will evaluate an effect of PKD2 on CCE by introducing dominant negative constructs of PKD2 in cell lines that endogenously express PKD1, PKD2 and TRPC1 and testing whether wild type or mutant PKD2 can regulate cAMP accumulation in kidney epithelial cells. Our ultimate goal is to develop a biologically significant system that would allow us to probe the mechanisms by which pathogenic mutations in PKD2 alter its normal function, and to design therapeutic interventions in diseases such as ADPKD.

Naturally occurring mutations in two separate genes, PKD1 and PKD2, are responsible for the vast majority (~99%) of all cases of autosomal dominant polycystic kidney disease (ADPKD), one of the most common genetic diseases affecting 1 in 1000 Americans. The hallmark of ADPKD is the development of epithelial cysts in the kidney, liver, and pancreas. Currently, there is no effective treatment for ADPKD. PKD1 encodes a large plasma membrane protein (PKD1 or Polycystin 1) with a long extracellular domain and has been speculated that it can function as an atypical G protein coupled receptor. PKD2 encodes an ion channel of the Transient Receptor Potential superfamily (TRPP2, PKD2, or Polycystin 2). However, the molecular function of these proteins and the mechanism(s) by which mutations in PKD1 and PKD2 cause ADPKD have been elusive. We have shown recently that PKD1 and TRPP2 form a complex at the plasma membrane that is activated by secreted WNT ligands. WNT proteins bind directly to the extracellular domain of PKD1 and induce Ca2+ influx and whole cell currents that are dependent on TRPP2. The PKD1/TRPP2 complex contains Dishevelleds (DVLs), which are cytoplasmic proteins that mediate Wnt signaling. The PKD1/TRPP2 complex has an essential role in directed cell migration and chemotaxis in response to a WNT ligand. In frog embryos pkd1 works together with wnt9a and dvl2 to control kidney tubular diameter. Therefore, we hypothesize that PKD1 and TRPP2 mediate WNT-induced Ca2+ signaling that is essential for directed cell migration and contributes to the determination of kidney tubule diameter. In this proposal, we will determine the mechanism of WNT- induced activation of PKD1/TRPP2 (Specific Aim 1). Determine the step(s) in WNT-induced directed cell migration specifically affected by PKD1 and TRPP2 (Specific Aim 2). Determine whether DVLs alone or in association with other cytosolic proteins linked to Wnt signaling function downstream of PKD1 and TRPP2 in WNT-induced cell migration (Specific Aim 3). This proposal is expected to shed light onto the mechanisms of WNT-induced activation of the PKD1/TRPP2, the mechanisms by which these proteins regulate directed cell migration, and cellular pathways activated immediately downstream of WNT-induced PKD1/TRPP2-mediated Ca2+ signaling. Knowledge of these pathways can be used as the springboard for the discovery of new druggable targets for ADPKD.

DESCRIPTION (provided by applicant): Healthy bone maintains a balance of bone formation mediated by osteoblasts and bone resorption mediated by osteoclasts. Many disease states, including chronic periodontitis, osteoporosis, rheumatoid arthritis, Paget's disease, and cancer metastases develop when osteoclasts are excessively recruited or inappropriately activated. Osteoclasts are constantly made throughout life from hematopoietic stem cells residing in the bone marrow through a series of complex events involving cytokine signaling and the microenvironment. Ca2+ signaling has an essential role in the regulation of osteoclastogenesis. Ca2+ channels activated in response to the depletion of intracellular Ca2+ stores have been suggested to mediate Ca2+ signaling in early stages of osteoclast formation. However, the exact molecules and the mechanism by which these channels control Ca2+ signaling in osteoclastogenesis are largely unknown. Using a combination of molecular, cell biological and whole animal studies, we show that the Transient Receptor Potential channel, TRPC1, enhances osteoclastogenesis at an early stage, whereas its inhibitor, the small cytosolic protein, I-mfa has an opposite effect. Enhanced osteoclastogenesis in I-mfa-null mice is corrected in mice lacking both genes indicating that TRPC1-mediated Ca2+ signaling has a dominant effect over I-mfa in osteoclast formation. Therefore, we propose that TRPC1 and I-mfa are essential for osteoclastogenesis by regulating Ca2+ signaling. This hypothesis will be tested by an integrated approach at the molecular, biophysical, cellular, and organismal levels by asking whether and how TRPC1 and I-mfa affect proliferation and priming of early osteoclast progenitors (Specific Aim 1), how TRPC1 and I-mfa modulate Ca2+ signaling in osteoclasts (Specific Aims 2 and 3), and whether TRPC1 and I-mfa affect osteoclastogenesis in a cell-autonomous fashion in vivo and in vitro and further, whether they affect osteoclast recruitment in experimentally induced animal models of osteoclastogenesis (Specific Aim 4). Our studies will lead to further understanding of critical pathways in the regulation of osteoclast development and function, which is needed to identify and develop new therapeutic interventions to control osteoclastogenesis and prevent bone loss.

Primary hyperparathyroidism is the one of the most common endocrine disorders. Primary hyperparathyroidism results from parathyroid adenomas while secondary hyperparathyroidism results from parathyroid gland hyperplasia in the setting of renal disease. Both conditions are associated with decreased expression of the Ca2+ sensing receptor (CaSR) which is required to suppress parathyroid hormone (PTH) secretion in the setting of hypercalcemia. Understanding the molecular mechanisms that regulate PTH secretion downstream of CaSR is critical for the discovery of new therapeutics to treat hyperparathyroidism and its associated morbidity. Here we show for the first time that Tprc1-null mice develop primary hyperparathyroidism, hypercalcemia, and low urinary calcium excretion mimicking the human disease Familial Hypocalciuric Hypercalcemia (FHH). FHH is a form of primary hypeparathyroidism and caused by inactivating mutations in CASR, GNA11 and AP2S1 encoding CaSR, the ?11 subunit of the guanine nucleotide-binding protein, and the ?1 subunit of the AP2 clathrin-associated adaptor complex mediating recycling of cell surface receptors and channels, respectively. Thus, we tested whether TRCP1 function is directly linked to CaSR signaling. Biochemical, functional, and cell biological experiments in vitro show that TRPC1 is activated by CaSR involving the action of G?11. We also show that TRPC1 physically interacts with AP2?1. These data lead us to the hypothesis that TRPC1 is required for normal suppression of PTH secretion in the parathyroid gland by acting downstream of CaSR via G?11. The interaction of TRPC1 with AP2?1 increases the availability of TRPC1 for CaSR-induced signaling by accelerating recycling of inactivated TRPC1. In Aim 1, we will employ cell biological approaches to define the mechanism by which TRPC1 mediates CaSR-induced Ca2+ signaling and the role of G?11 and AP2?1 in this signaling pathway in cells derived from the parathyroid gland. In specific Aim 2, we will determine whether TRPC1 functions downstream of CaSR and G?11 in vivo, by asking whether compound mice lacking Trpc1 and Casr or Gn?11/Gn?q genes in their parathyroid glands show more severe FHH-like phenotypes than phenotypes elicited by single gene deletions. Understanding the pathways that regulate PTH secretion could have a high impact on designing new and more effective and specific approaches to treat patients with primary as well as secondary hyperparathyroidism.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common genetic diseases affecting 12.5 million people worldwide and by far, the most common genetic disease of the kidney. It is caused by inactivating mutations in the PKD1 or PKD2 genes, encoding a receptor-channel complex (Polycystins or PKD1/PKD2). A hallmark of ADPKD is increased cell proliferation. However, how mutations in the Polycystin genes cause increased cell proliferation is not completely understood. A key organelle in disease development and progression is the primary cilium, an antenna-like organelle housing several mitogenic signaling pathways. Genetic and pharmacologic studies show that primary cilia ablation or acceleration of cilia disassembly reduces cell proliferation, suppresses cystic growth and improves kidney function, whereas deceleration of ciliary disassembly has the opposite effects in mouse models of ADPKD. While these observations are of paramount importance in understanding the pathophysiology of ADPKD and in developing therapeutic approaches to slow down disease progression, a unifying theory connecting cilia and cell proliferation in ADPKD is lacking. Ciliary assembly and disassembly or shedding are normal processes of actively proliferating cells. Cilia assemble in quiescent cells, while disassemble or shed when cells re-enter the cell cycle (G1/S). Our preliminary data show that deletion of Pkd1 increases the activity/levels of p53, which in turn, induces the expression of the substrate recognition receptor FBW7 of the SCFFBW7 Ubiquitin E3 ligase. FBW7 targets for proteasomal degradation a subset of disassembly factors delaying deciliation and stabilizing primary cilia. Continuous presence of cilia during the G1/S transition leads to sustained mitogenic signaling mediated by stabilized/remaining cilia resulting in more cells eventually entering the cell cycle. Finally, genetic modifications of this pathway, improve renal function of Pkd1-null mice. These results help explain the increased cell proliferation seen in ADPKD kidneys and the positive effect of cilia on disease progression. Using a vertical approach combining biochemical, cell biological, and genetic methods, we will determine the role of ciliary disassembly and shedding in cystic kidney disease progression. Successful completion of the proposed will have a significant impact on our understanding the biological role of ciliary disassembly/shedding in disease progression and on helping develop new therapeutic approaches for ADPKD.