Ttc21b deficiency attenuates autosomal dominant polycystic kidney disease in a kidney tubular- and maturation-dependent manner.

Primary cilia are sensory organelles built and maintained by intraflagellar transport (IFT) multiprotein complexes. Deletion of several IFT-B genes attenuates polycystic kidney disease (PKD) severity in juvenile and adult autosomal dominant polycystic kidney disease (ADPKD) mouse models. However, deletion of an IFT-A adaptor, Tulp3, attenuates PKD severity in adult mice only. These studies indicate that dysfunction of specific cilia components has potential therapeutic value. To broaden our understanding of cilia dysfunction and its therapeutic potential, we investigate the role of global deletion of an IFT-A gene, Ttc21b, in juvenile and adult mouse models of ADPKD. Both juvenile (postnatal day 21) and adult (six months of age) ADPKD mice exhibited kidney cysts, increased kidney weight/body weight ratios, lengthened kidney cilia, inflammation, and increased levels of the nutrient sensor, O-linked ß-N-acetylglucosamine (O-GlcNAc). Deletion of Ttc21b in juvenile ADPKD mice reduced cortical collecting duct cystogenesis and kidney weight/body weight ratios, increased proximal tubular and glomerular dilations, but did not reduce cilia length, inflammation, nor O-GlcNAc levels. In contrast, Ttc21b deletion in adult ADPKD mice markedly attenuated kidney cystogenesis and reduced cilia length, inflammation, and O-GlcNAc levels. Thus, unlike IFT-B, the effect of Ttc21b deletion in mouse models of ADPKD is development-specific. Unlike an IFT-A adaptor, deleting Ttc21b in juvenile ADPKD mice is partially ameliorative. Thus, our studies suggest that different microenvironmental factors, found in distinct nephron segments and in developing versus mature stages, modify ciliary homeostasis and ADPKD pathobiology. Further, elevated levels of O-GlcNAc, which regulates cellular metabolism and ciliogenesis, may be a pathological feature of ADPKD.

Thm2 interacts with paralog, Thm1, and sensitizes to Hedgehog signaling in postnatal skeletogenesis.

Mutations in the intraflagellar transport-A (IFT-A) gene, THM1, have been identified in skeletal ciliopathies. Here, we report a genetic interaction between Thm1, and its paralog, Thm2, in postnatal skeletogenesis. THM2 localizes to primary cilia, but Thm2 deficiency does not affect ciliogenesis and Thm2-null mice survive into adulthood. However, by postnatal day 14, Thm2-/-; Thm1aln/+ mice exhibit small stature and small mandible. Radiography and microcomputed tomography reveal Thm2-/-; Thm1aln/+ tibia are less opaque and have reduced cortical and trabecular bone mineral density. In the mutant tibial growth plate, the proliferation zone is expanded and the hypertrophic zone is diminished, indicating impaired chondrocyte differentiation. Additionally, mutant growth plate chondrocytes show increased Hedgehog signaling. Yet deletion of one allele of Gli2, a major transcriptional activator of the Hedgehog pathway, exacerbated the Thm2-/-; Thm1aln/+ small phenotype, and further revealed that Thm2-/-; Gli2+/- mice have small stature. In Thm2-/-; Thm1aln/+ primary osteoblasts, a Hedgehog signaling defect was not detected, but bone nodule formation was markedly impaired. This indicates a signaling pathway is altered, and we propose that this pathway may potentially interact with Gli2. Together, our data reveal that loss of Thm2 with one allele of Thm1, Gli2, or both, present new IFT mouse models of osteochondrodysplasia. Our data also suggest Thm2 as a modifier of Hedgehog signaling in postnatal skeletal development.

Intraflagellar-transport A dysfunction causes hyperphagia-induced systemic insulin resistance in a pre-obese state.

Deletion of murine Thm1, an intraflagellar transport A (IFT-A) component that mediates ciliary protein trafficking, causes hyperphagia, obesity, and metabolic syndrome. The role of Thm1 or IFT-A in adipogenesis and insulin sensitivity is unknown. Here, we report that Thm1 knockdown in 3T3-L1 pre-adipocytes promotes adipogenesis and enhances insulin sensitivity in vitro. Yet, pre-obese Thm1 conditional knockout mice show systemic insulin resistance. While insulin-induced AKT activation in Thm1 mutant adipose depots and skeletal muscle are similar to those of control littermates, an attenuated insulin response arises in the mutant liver. Insulin treatment of control and Thm1 mutant primary hepatocytes results in similar AKT activation. Moreover, pair-feeding Thm1 conditional knockout mice produces a normal insulin response, both in the liver and systemically. Thus, hyperphagia caused by a cilia defect, induces hepatic insulin resistance via a non-cell autonomous mechanism. In turn, hepatic insulin resistance drives systemic insulin resistance prior to an obese phenotype. These data demonstrate that insulin signaling across cell types is regulated differentially, and that the liver is particularly susceptible to hyperphagia-induced insulin resistance and a critical determinant of systemic insulin resistance.

Analysis of primary cilia in renal tissue and cells.

Primary cilia are singular, sensory organelles that extend from the plasma membrane of most quiescent mammalian cells. These slender, microtubule-based organelles receive and transduce extracellular cues and regulate signaling pathways. Primary cilia are critical to the development and function of many tissue types, and mutation of ciliary genes causes multi-system disorders, termed ciliopathies. Notably, renal cystic disease is one of the most common clinical features of ciliopathies, highlighting a central role for primary cilia in the kidney. Additionally, acute kidney injury and chronic kidney disease are associated with altered primary cilia lengths on renal epithelial cells, suggesting ciliary dynamics and renal physiology are linked. Here we describe methods to examine primary cilia in kidney tissue and in cultured renal cells. We include immunofluorescence and scanning electron microscopy to determine ciliary localization of proteins and cilia structure. Further, we detail cellular assays to measure cilia assembly and disassembly, which regulate cilia length.

Inhibition of Hedgehog signaling suppresses proliferation and microcyst formation of human Autosomal Dominant Polycystic Kidney Disease cells.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is caused by mutation of PKD1 or PKD2, which encode polycystin 1 and 2, respectively. The polycystins localize to primary cilia and the functional loss of the polycystin complex leads to the formation and progressive growth of fluid-filled cysts in the kidney. The pathogenesis of ADPKD is complex and molecular mechanisms connecting ciliary dysfunction to renal cystogenesis are unclear. Primary cilia mediate Hedgehog signaling, which modulates cell proliferation and differentiation in a tissue-dependent manner. Previously, we showed that Hedgehog signaling was increased in cystic kidneys of several PKD mouse models and that Hedgehog inhibition prevented cyst formation in embryonic PKD mouse kidneys treated with cAMP. Here, we show that in human ADPKD tissue, Hedgehog target and activator, Glioma 1, was elevated and localized to cyst-lining epithelial cells and to interstitial cells, suggesting increased autocrine and paracrine Hedgehog signaling in ADPKD, respectively. Further, Hedgehog inhibitors reduced basal and cAMP-induced proliferation of ADPKD cells and cyst formation in vitro. These data suggest that Hedgehog signaling is increased in human ADPKD and that suppression of Hedgehog signaling can counter cellular processes that promote cyst growth in vitro.

Dysfunction of intraflagellar transport-A causes hyperphagia-induced obesity and metabolic syndrome.

Primary cilia extend from the plasma membrane of most vertebrate cells and mediate signaling pathways. Ciliary dysfunction underlies ciliopathies, which are genetic syndromes that manifest multiple clinical features, including renal cystic disease and obesity. THM1 (also termed TTC21B or IFT139) encodes a component of the intraflagellar transport-A complex and mutations in THM1 have been identified in 5% of individuals with ciliopathies. Consistent with this, deletion of murine Thm1 during late embryonic development results in cystic kidney disease. Here, we report that deletion of murine Thm1 during adulthood results in obesity, diabetes, hypertension and fatty liver disease, with gender differences in susceptibility to weight gain and metabolic dysfunction. Pair-feeding of Thm1 conditional knock-out mice relative to control littermates prevented the obesity and related disorders, indicating that hyperphagia caused the obese phenotype. Thm1 ablation resulted in increased localization of adenylyl cyclase III in primary cilia that were shortened, with bulbous distal tips on neurons of the hypothalamic arcuate nucleus, an integrative center for signals that regulate feeding and activity. In pre-obese Thm1 conditional knock-out mice, expression of anorexogenic pro-opiomelanocortin (Pomc) was decreased by 50% in the arcuate nucleus, which likely caused the hyperphagia. Fasting of Thm1 conditional knock-out mice did not alter Pomc nor orexogenic agouti-related neuropeptide (Agrp) expression, suggesting impaired sensing of changes in peripheral signals. Together, these data indicate that the Thm1-mutant ciliary defect diminishes sensitivity to feeding signals, which alters appetite regulation and leads to hyperphagia, obesity and metabolic disease.

Downregulating hedgehog signaling reduces renal cystogenic potential of mouse models.

Renal cystic diseases are a leading cause of renal failure. Mutations associated with renal cystic diseases reside in genes encoding proteins that localize to primary cilia. These cystoproteins can disrupt ciliary structure or cilia-mediated signaling, although molecular mechanisms connecting cilia function to renal cystogenesis remain unclear. The ciliary gene, Thm1(Ttc21b), negatively regulates Hedgehog signaling and is most commonly mutated in ciliopathies. We report that loss of murine Thm1 causes cystic kidney disease, with persistent proliferation of renal cells, elevated cAMP levels, and enhanced expression of Hedgehog signaling genes. Notably, the cAMP-mediated cystogenic potential of Thm1-null kidney explants was reduced by genetically deleting Gli2, a major transcriptional activator of the Hedgehog pathway, or by culturing with small molecule Hedgehog inhibitors. These Hedgehog inhibitors acted independently of protein kinase A and Wnt inhibitors. Furthermore, simultaneous deletion of Gli2 attenuated the renal cystic disease associated with deletion of Thm1. Finally, transcripts of Hedgehog target genes increased in cystic kidneys of two other orthologous mouse mutants, jck and Pkd1, and Hedgehog inhibitors reduced cystogenesis in jck and Pkd1 cultured kidneys. Thus, enhanced Hedgehog activity may have a general role in renal cystogenesis and thereby present a novel therapeutic target.

Myosin-X functions in polarized epithelial cells.

Myosin-X (Myo10) is an unconventional myosin that localizes to the tips of filopodia and has critical functions in filopodia. Although Myo10 has been studied primarily in nonpolarized, fibroblast-like cells, Myo10 is expressed in vivo in many epithelia-rich tissues, such as kidney. In this study, we investigate the localization and functions of Myo10 in polarized epithelial cells, using Madin-Darby canine kidney II cells as a model system. Calcium-switch experiments demonstrate that, during junction assembly, green fluorescent protein-Myo10 localizes to lateral membrane cell-cell contacts and to filopodia-like structures imaged by total internal reflection fluorescence on the basal surface. Knockdown of Myo10 leads to delayed recruitment of E-cadherin and ZO-1 to junctions, as well as a delay in tight junction barrier formation, as indicated by a delay in the development of peak transepithelial electrical resistance (TER). Although Myo10 knockdown cells eventually mature into monolayers with normal TER, these monolayers do exhibit increased paracellular permeability to fluorescent dextrans. Importantly, knockdown of Myo10 leads to mitotic spindle misorientation, and in three-dimensional culture, Myo10 knockdown cysts exhibit defects in lumen formation. Together these results reveal that Myo10 functions in polarized epithelial cells in junction formation, regulation of paracellular permeability, and epithelial morphogenesis.

Intracellular Distribution-based Anticancer Drug Targeting: Exploiting a Lysosomal Acidification Defect Associated with Cancer Cells.

The therapeutic usefulness of anticancer agents relies on their ability to exert maximal toxicity to cancer cells and minimal toxicity to normal cells. The difference between these two parameters defines the therapeutic index of the agent. Towards this end, much research has focused on the design of anticancer agents that have optimized potency against a variety of cancer cell types; however, much less effort is spent on the design of drugs that are minimally toxic to normal cells. We have previously described a concept for a novel drug delivery platform that relies on the propensity of drugs with optimal physicochemical properties to distribute differently in normal versus cancer cells due to differences in intracellular pH gradients. Specifically, we demonstrated in vitro that certain weakly basic anticancer agents had the propensity to distribute to intracellular locations in normal cells that prevent interaction with the drug target, and to intracellular locations in cancer cells that promote drug-target interactions. We refer to this concept broadly as intracellular distribution-based drug targeting. Here we will discuss current in vivo work from our laboratory that examined the role of lysosome pH on the intracellular distribution and toxicity of inhibitors of the Hsp90 molecular chaperone in mice.

Myosin Vc is a molecular motor that functions in secretory granule trafficking.

Class V myosins are actin-based motor proteins that have critical functions in organelle trafficking. Of the three class V myosins expressed in mammals, relatively little is known about Myo5c except that it is abundant in exocrine tissues. Here we use MCF-7 cells to identify the organelles that Myo5c associates with, image the dynamics of Myo5c in living cells, and test the functions of Myo5c. Endogenous Myo5c localizes to two distinct compartments: small puncta and slender tubules. Myo5c often exhibits a highly polarized distribution toward the leading edge in migrating cells and is clearly distinct from the Myo5a or Myo5b compartments. Imaging with GFP-Myo5c reveals that Myo5c puncta move slowly (approximately 30 nm/s) and microtubule independently, whereas tubules move rapidly (approximately 440 nm/s) and microtubule dependently. Myo5c puncta colocalize with secretory granule markers such as chromogranin A and Rab27b, whereas Myo5c tubules are labeled by Rab8a. TIRF imaging indicates that the granules can be triggered to undergo secretion. To test if Myo5c functions in granule trafficking, we used the Myo5c tail as a dominant negative and found that it dramatically perturbs the distribution of granule markers. These results provide the first live-cell imaging of Myo5c and indicate that Myo5c functions in secretory granule trafficking.

A novel form of motility in filopodia revealed by imaging myosin-X at the single-molecule level.

Although many proteins, receptors, and viruses are transported rearward along filopodia by retrograde actin flow, it is less clear how molecules move forward in filopodia. Myosin-X (Myo10) is an actin-based motor hypothesized to use its motor activity to move forward along actin filaments to the tips of filopodia. Here we use a sensitive total internal reflection fluorescence (TIRF) microscopy system to directly visualize the movements of GFP-Myo10. This reveals a novel form of motility at or near the single-molecule level in living cells wherein extremely faint particles of Myo10 move in a rapid and directed fashion toward the filopodial tip. These fast forward movements occur at approximately 600 nm/s over distances of up to approximately 10 microm and require Myo10 motor activity and actin filaments. As expected for imaging at the single-molecule level, the faint particles of GFP-Myo10 are diffraction limited, have an intensity range similar to single GFP molecules, and exhibit stepwise bleaching. Faint particles of GFP-Myo5a can also move toward the filopodial tip, but at a slower characteristic velocity of approximately 250 nm/s. Similar movements were not detected with GFP-Myo1a, indicating that not all myosins are capable of intrafilopodial motility. These data indicate the existence of a novel system of long-range transport based on the rapid movement of myosin molecules along filopodial actin filaments.

Transduced viral IL-10 is exocytosed from lacrimal acinar secretory vesicles in a myosin-dependent manner in response to carbachol.

The purpose of this study was to determine the intracellular trafficking and release pathways for the therapeutic protein, viral IL-10 (vIL-10), from transduced acinar epithelial cells from rabbit lacrimal gland. Primary cultured rabbit lacrimal gland acinar cells (LGACs) were transduced with adenovirus serotype 5 containing viral interleukin-10 (AdvIL-10). The distribution of vIL-10 was assessed by confocal fluorescence microscopy. Carbachol (CCH)-stimulated release of vIL-10 was quantified by ELISA. vIL-10 localization and exocytosis was probed in response to treatments with agents modulating actin- and myosin-based transport. vIL-10 immunoreactivity was detected in large intracellular vesicles in transduced LGAC. vIL-10 was partially co-localized with biosynthetic but not endosomal compartment markers. vIL-10 release was sensitive to CCH, and the kinetics of release showed an initial burst phase that was similar but not identical to that of the secretory protein, beta-hexosaminidase. Disassembly of actin filaments with latrunculin B significantly increased CCH-stimulated vIL-10 secretion, suggesting that vIL-10 was released from stores sequestered beneath the subapical actin barrier. That release required the activity of actin-dependent myosin motors previously implicated in secretory vesicle exocytosis was confirmed by findings that CCH-stimulated vIL-10 release was reduced by inhibition of non-muscle myosin 2 and myosin 5c function, using ML-7 and overexpression of dominant negative myosin 5c, respectively. These results suggest that the majority of vIL-10 transgene product is packaged into a subpopulation of secretory vesicles that utilize actin-dependent myosin motors for aspects of actin coat assembly, compound fusion and exocytosis at the apical plasma membrane in response to CCH stimulation.

The class V myosin motor, myosin 5c, localizes to mature secretory vesicles and facilitates exocytosis in lacrimal acini.

We investigated the role of the actin-based myosin motor, myosin 5c (Myo5c) in vesicle transport in exocrine secretion. Lacrimal gland acinar cells (LGAC) are the major source for the regulated secretion of proteins from the lacrimal gland into the tear film. Confocal fluorescence and immunogold electron microscopy revealed that Myo5c was associated with secretory vesicles in primary rabbit LGAC. Upon stimulation of secretion with the muscarinic agonist, carbachol, Myo5c was also detected in association with actin-coated fusion intermediates. Adenovirus-mediated expression of green fluorescent protein (GFP) fused to the tail domain of Myo5c (Ad-GFP-Myo5c-tail) showed that this protein was localized to secretory vesicles. Furthermore, its expression induced a significant (P < or = 0.05) decrease in carbachol-stimulated release of two secretory vesicle content markers, secretory component and syncollin-GFP. Adenovirus-mediated expression of GFP appended to the full-length Myo5c (Ad-GFP-Myo5c-full) was used in parallel with adenovirus-mediated expression of GFP-Myo5c-tail in LGAC to compare various parameters of secretory vesicles labeled with either GFP-labeled protein in resting and stimulated LGAC. These studies revealed that the carbachol-stimulated increase in secretory vesicle diameter associated with compound fusion of secretory vesicles that was also exhibited by vesicles labeled with GFP-Myo5c-full was impaired in vesicles labeled with GFP-Myo5c-tail. A significant decrease in GFP labeling of actin-coated fusion intermediates was also seen in carbachol-stimulated LGAC transduced with GFP-Myo5c-tail relative to LGAC transduced with GFP-Myo5c-full. These results suggest that Myo5c participates in apical exocytosis of secretory vesicles.