Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis.

There is significant interest in the use of primary intestinal epithelial cells in monolayer culture to model intestinal biology. However, it has proven to be challenging to create functional, differentiated monolayers using current culture methods, likely due to the difficulty in expanding these cells. Here, we adapted our recently developed method for the culture of intestinal epithelial spheroids to establish primary epithelial cell monolayers from the colon of multiple genetic mouse strains. These monolayers contained differentiated epithelial cells that displayed robust transepithelial electrical resistance. We then functionally tested them by examining immunoglobulin A (IgA) transcytosis across Transwells. IgA transcytosis required induction of polymeric Ig receptor (pIgR) expression, which could be stimulated by a combination of lipopolysaccharide and inhibition of γ-secretase. In agreement with previous studies using immortalized cell lines, we found that tumor necrosis factor-α, interleukin (IL)-1ß, IL-17, and heat-killed microbes also stimulated pIgR expression and IgA transcytosis. We used wild-type and knockout cells to establish that among these cytokines, IL-17 was the most potent inducer of pIgR expression/IgA transcytosis. Interferon-γ, however, did not induce pIgR expression, and instead led to cell death. This new method will allow the use of primary cells for studies of intestinal physiology.

The role of autophagy in Paneth cell differentiation and secretion.

Paneth cells are a small intestinal epithelial cell lineage that is considered to have a role in innate immune function. Recent studies on mice with diminished and/or loss of autophagy have suggested that Paneth cells are a primary target in vivo. Interestingly, loss of autophagy affects the secretion of antimicrobial proteins from Paneth cells. Understanding the intersection of the autophagy pathway with the secretory apparatus, which is a key feature of differentiation of Paneth cells, is a key unanswered question.

The stem cell niche.

Virtually every tissue of the adult organism maintains a population of putatively slowly-cycling stem cells that maintain homeostasis of the tissue and respond to injury when challenged. These cells are regulated and supported by the surrounding microenvironment, referred to as the stem cell 'niche'. The niche includes all cellular and non-cellular components that interact in order to control the adult stem cell, and these interactions can often be broken down into one of two major mechanistic categories--physical contact and diffusible factors. The niche has been studied directly and indirectly in a number of adult stem cell systems. Herein, we will first focus on the most well-understood niches supporting the germline stem cells in the lower organisms Caenorhabditis elegans and Drosophila melanogaster before concentrating on the more complex, less well-understood mammalian niches supporting the neural, epidermal, haematopoietic and intestinal stem cells.

Extranuclear sequestration of phospho-Jun N-terminal kinase and distorted villi produced by activated Rac1 in the intestinal epithelium of chimeric mice.

Previously, we used a genetic mosaic system to conduct an in vivo analysis of the effects of Rac1 activation on the developing intestinal epithelium ( Stappenbeck, T. S. and Gordon, J. I. (2000) Development 127, 2629-2642). Expression of a constitutively active human Rac1 (Rac1Leu61) in the 129/Sv-derived small intestinal epithelium of C57Bl/6-ROSA26<-->129/Sv chimeric mice led to precocious differentiation of some lineages with accompanying alterations in their apical actin. We have now explored the underlying mechanisms. Rac1Leu61 leads to accumulation of the 46 kDa form of phosphorylated Jun N-terminal kinase (p-Jnk) in the apical cytoplasm, but not in the nucleus of E18.5 proliferating and differentiating intestinal epithelial cells. The effect is cell-autonomous, selective for this mitogen-activated protein kinase family member, and accompanied by apical cytoplasmic accumulation of p21-activated kinase. c-Jun, a downstream nuclear target of p-Jnk, does not show evidence of enhanced phosphorylation, providing functional evidence for cytoplasmic sequestration of p-Jnk in Rac1Leu61-expressing epithelium. In adult chimeras, Rac1 activation augments cell proliferation in crypts of Lieberkühn, without a compensatory change in basal apoptosis and produces a dramatic, very unusual widening of villi. These results reveal a novel in vivo paradigm for Rac1 activation involving p-Jnk-mediated signaling at a distinctive extra-nuclear site, with associated alterations in the actin cytoskeleton. They also provide a new perspective about the determinants of small intestinal villus morphogenesis.

Immune system dysfunction and autoimmune disease in mice lacking Emk (Par-1) protein kinase.

Emk is a serine/threonine protein kinase implicated in regulating polarity, cell cycle progression, and microtubule dynamics. To delineate the role of Emk in development and adult tissues, mice lacking Emk were generated by targeted gene disruption. Emk(-/-) mice displayed growth retardation and immune cell dysfunction. Although B- and T-cell development were normal, CD4(+)T cells lacking Emk exhibited a marked upregulation of the memory marker CD44/pgp-1 and produced more gamma interferon and interleukin-4 on stimulation through the T-cell receptor in vitro. In addition, B-cell responses to T-cell-dependent and -independent antigen challenge were altered in vivo. As Emk(-/-) animals aged, they developed splenomegaly, lymphadenopathy, membranoproliferative glomerulonephritis, and lymphocytic infiltrates in the lungs, parotid glands and kidneys. Taken together, these results demonstrate that the Emk protein kinase is essential for maintaining immune system homeostasis and that loss of Emk may contribute to autoimmune disease in mammals.

Genetic mosaic analysis based on Cre recombinase and navigated laser capture microdissection.

Defining molecular interactions that occur at the interface between "normal" and "abnormal" cell populations represents an important but often underexplored aspect of the pathogenesis of diseases with focal origins. Here, we illustrate an approach for conducting such analyses based on mosaic patterns of Cre recombinase expression in the adult mouse intestinal epithelium. Transgenic mice were generated that express Cre in the stem cell niche of crypts located in specified regions of their intestine. Some of these mice were engineered to allow for doxycycline-inducible Cre expression. Recombination in all pedigrees was mosaic: Cre-expressing crypts that supported recombination in all of their active multipotent stem cells were located adjacent to "control" crypts that did not express Cre at detectable levels. Cre-mediated recombination of a floxed LacZ reporter provided direct evidence that adult small-intestinal crypts contain more than one active multipotent stem cell, and that these cells can be retained in both small-intestinal and colonic crypts for at least 80 d. A method was developed to recover epithelial cells from crypts with or without recombination for subsequent gene expression profiling. Stained sections of intestine were used to create electronic image templates to guide laser capture microdissection (LCM) of adjacent frozen sections. This navigated form of LCM overcomes problems with mRNA degradation encountered when cells are marked directly by immunohistochemical methods. Combining Cre-engineered genetic mosaic mice with navigated-LCM will allow biology and pathobiology to be explored at the junction between normal and perturbed cellular cohorts.

Rac1 mutations produce aberrant epithelial differentiation in the developing and adult mouse small intestine.

The mouse small intestinal epithelium undergoes continuous renewal throughout life. Previous studies suggest that differentiation of this epithelium is regulated by instructions that are received as cells migrate along crypt-villus units. The nature of the instructions and their intracellular processing remain largely undefined. In this report, we have used genetic mosaic analysis to examine the role of Rac1 GTPase-mediated signaling in controlling differentiation. A constitutively active mutation (Rac1Leu61) or a dominant negative mutation (Rac1Asn17) was expressed in the 129/Sv embryonic stem cell-derived component of the small intestine of C57Bl/6-ROSA26<->129/Sv mice. Rac1Leu61 induces precocious differentiation of members of the Paneth cell and enterocytic lineages in the proliferative compartment of the fetal gut, without suppressing cell division. Forced expression of the dominant negative mutation inhibits epithelial differentiation, without affecting cell division, and slows enterocytic migration along crypt-villus units. The effects produced by Rac1Leu61 or Rac1Asn17 in the 129/Sv epithelium do not spread to adjacent normal C57Bl/6 epithelial cells. These results provide in vivo evidence that Rac1 is involved in the import and intracellular processing of signals that control differentiation of a mammalian epithelium.

Notes from some crypt watchers: regulation of renewal in the mouse intestinal epithelium.

The mouse intestinal epithelium undergoes rapid renewal throughout life, thereby requiring continuous coordination of its cellular proliferation, differentiation, and death programs. Recent advances in our understanding of this process have highlighted some of the molecules that regulate renewal and their potential roles in gut neoplasia.

Breaking the connection: displacement of the desmosomal plaque protein desmoplakin from cell-cell interfaces disrupts anchorage of intermediate filament bundles and alters intercellular junction assembly.

The desmosomal plaque protein desmoplakin (DP), located at the juncture between the intermediate filament (IF) network and the cytoplasmic tails of the transmembrane desmosomal cadherins, has been proposed to link IF to the desmosomal plaque. Consistent with this hypothesis, previous studies of individual DP domains indicated that the DP COOH terminus associates with IF networks whereas NH2-terminal sequences govern the association of DP with the desmosomal plaque. Nevertheless, it had not yet been demonstrated that DP is required for attaching IF to the desmosome. To test this proposal directly, we generated A431 cell lines stably expressing DP NH2-terminal polypeptides, which were expected to compete with endogenous DP during desmosome assembly. As these polypeptides lacked the COOH-terminal IF-binding domain, this competition should result in the loss of IF anchorage if DP is required for linking IF to the desmosomal plaque. In such cells, a 70-kD DP NH2-terminal polypeptide (DP-NTP) colocalized at cell-cell interfaces with desmosomal proteins. As predicted, the distribution of endogenous DP was severely perturbed. At cell-cell borders where endogenous DP was undetectable by immunofluorescence, there was a striking absence of attached tonofibrils (IF bundles). Furthermore, DP-NTP assembled into ultrastructurally identifiable junctional structures lacking associated IF bundles. Surprisingly, immunofluorescence and immunogold electron microscopy indicated that adherens junction components were coassembled into these structures along with desmosomal components and DP-NTP. These results indicate that DP is required for anchoring IF networks to desmosomes and furthermore suggest that the DP-IF complex is important for governing the normal spatial segregation of adhesive junction components during their assembly into distinct structures.

Phosphorylation of the desmoplakin COOH terminus negatively regulates its interaction with keratin intermediate filament networks.

Desmoplakins (DPs) are the most abundant proteins in the innermost portion of the desmosomal plaque and have been proposed to play a role in the attachment of intermediate filaments (IF) to cell-cell contact sites. Our previous results suggest that the globular end domains of DP perform dual functions: first, to target DP to the desmosome via the NH2 terminus and second, to attach IF to the desmosomal plaque via the COOH terminus. When ectopically expressed in most cultured cells, the COOH terminus plus the rod domain (DP. delta N.SerC23) exhibits striking coalignment with keratin IF networks. However, in certain cell types (e.g. PtK2) or in cells treated with forskolin to activate protein kinase A, DP. delta N.SerC23 exhibits a diffuse cytoplasmic distribution. A variant molecule (DP. delta N.GlyC23) in which a serine located 23 amino acids from the COOH terminus is altered to a glycine, thereby disrupting a protein kinase A consensus phosphorylation site, co-localizes with keratin IF networks regardless of cell type or forskolin treatment. Analysis of the phosphopeptide maps of these DP variants and endogenous DP is consistent with the phosphorylation of the serine 23 residues from the COOH terminus. These results suggest that phosphorylation of a specific residue in the DP COOH terminus may negatively regulate its interaction with keratin IF networks.

Structure and function of desmosomal transmembrane core and plaque molecules.

Desmosomes are intercellular junctions that function in cell-cell adhesion and attachment of intermediate filaments (IF) to the cell surface. Desmogleins and desmocollins are the major components of the transmembrane adhesion complex, whereas desmoplakins (DPs) are the most prominent components of the cytoplasmic plaque. Based on sequence similarity, desmogleins and desmocollins are related to the calcium-dependent homophilic adhesion molecules known as cadherins. Like the classical cadherins, the desmosomal cadherins contain four homologous extracellular domains bearing putative calcium-binding sites, a single transmembrane spanning domain, and a C-terminal cytoplasmic tail. Molecules in the desmoglein subclass contain a unique C-terminal extension within which is found a repeating motif that is predicted to form two beta-strands and two turns. Stable cell lines expressing desmoglein 1 have been generated from normally non-adherent L cell fibroblasts, to study the contribution of this cadherin to desmosomal adhesion. The predicted sequence of desmoplakin (DP) I suggests it will form homodimers comprising a central alpha-helical coiled-coil rod and two globular end domains. The C-terminus contains three regions with significant homology, each of which is made up of a 38-residue motif also found in two other molecules involved in organization of IF, bullous pemphigoid antigen and plectin. Ectopically expressed polypeptides including the C-terminus of DP I specifically align with keratin and vimentin IF in cultured cells, whereas those lacking this domain do not align with IF. The last 68 amino acids of DP are required for alignment along keratin but not vimentin IF, and residues 48-68 from the C-terminal end are critical for this interaction. These results suggest that the C-terminus of DP plays a role in the attachment of IF to the desmosome and that a specific site is necessary for interaction with keratin IF. A sequence at the most N-terminal end of DP appears to be required for efficient incorporation into the desmosomal plaque. Interestingly, this region has not been reported to be present in the homologous bullous pemphigoid antigen or plectin molecules and may represent a desmosomal targeting sequence.

Functional analysis of desmoplakin domains: specification of the interaction with keratin versus vimentin intermediate filament networks.

We previously demonstrated that truncated desmoplakin I (DP I) molecules containing the carboxyl terminus specifically coalign with and disrupt both keratin and vimentin intermediate filament (IF) networks when overexpressed in tissue culture cells (Stappenbeck, T. S., and K. J. Green. J. Cell Biol. 116:1197-1209). These experiments suggested that the DP carboxyl-terminal domain is involved either directly or indirectly in linking IF with the desmosome. Using a similar approach, we have now investigated the behavior of ectopically expressed full-length DP I in cultured cells. In addition, we have further dissected the functional sequences in the carboxyl terminus of DP I that facilitate the interaction with IF networks. Transient transfection of a clone encoding full-length DP I into COS-7 cells produced protein that appeared in some cells to associate with desmosomes and in others to coalign with and disrupt IF. Deletion of the carboxyl terminus from this clone resulted in protein that still appeared capable of associating with desmosomes but not interacting with IF networks. As the amino terminus appeared to be dispensable for IF interaction, we made finer deletions in the carboxyl terminus of DP based on blocks of sequence similarity with the related molecules bullous pemphigoid antigen and plectin. We found a sequence at the very carboxyl terminus of DP that was necessary for coalignment with and disruption of keratin IF but not vimentin IF. Furthermore, the coalignment of specific DP proteins along keratin IF but not vimentin IF was correlated with resistance to extraction by Triton. The striking uncoupling resulting from the deletion of specific DP sequences suggests that the carboxyl terminus of DP interacts differentially with keratin and vimentin IF networks.

Structure of desmoplakin and its association with intermediate filaments.

Desmoplakins (DPs) I and II are two major related proteins located in the desmosomal plaque where they have been proposed to play a role in attaching intermediate filaments (IF) to the inner cell surface. The predicted amino acid sequence of DP was obtained by analysis of overlapping cDNA clones. Computer-aided analysis suggests that DPI will form a dumbbell-shaped homodimer, with a central alpha-helical coiled coil rod domain of 132 nm and two globular end domains. The DPII molecule is missing 599 residues from the central domain, resulting in a rod about one third the length of DPI. The carboxyl terminus comprises three subdomains each containing almost 5 repeats of a 38 residue repeating motif with a periodicity in acidic and basic residues similar to that found in the rod domain of IF proteins. This suggests a possible mechanism by which these proteins might interact. The amino terminus contains groups of heptad repeats that are predicted to form at least two major alpha-helical rich bundles. A series of c-myc-tagged mammalian expression vectors encoding specific predicted domains of DPI were transiently expressed in COS-7 cells. Light and electron microscopical observations revealed that DP polypeptides including the 90 kDa carboxyl terminal globular domain of DPI specifically colocalized with and ultimately resulted in the complete disruption of keratin and vimentin IF. This effect was specific for the carboxyl terminus, as the expression of the 95 kDa rod domain of DPI did not visibly alter IF networks.(ABSTRACT TRUNCATED AT 250 WORDS)

The desmoplakin carboxyl terminus coaligns with and specifically disrupts intermediate filament networks when expressed in cultured cells.

Specific interactions between desmoplakins I and II (DP I and II) and other desmosomal or cytoskeletal molecules have been difficult to determine in part because of the complexity and insolubility of the desmosome and its constituents. We have used a molecular genetic approach to investigate the role that DP I and II may play in the association of the desmosomal plaque with cytoplasmic intermediate filaments (IF). A series of mammalian expression vectors encoding specific predicted domains of DP I were transiently expressed in cultured cells that form (COS-7) and do not form (NIH-3T3) desmosomes. Sequence encoding a small antigenic peptide was added to the 3' end of each mutant DP cDNA to facilitate immunolocalization of mutant DP protein. Light and electron microscopical observations revealed that DP polypeptides including the 90-kD carboxy-terminal globular domain of DP I specifically colocalized with and ultimately resulted in the complete disruption of IF in both cell lines. This effect was specific for IF as microtubule and microfilament networks were unaltered. This effect was also specific for the carboxyl terminus of DP, as the expression of the 95-kD rod domain of DP I did not visibly alter IF networks. Immunogold localization of COS-7 cells transfected with constructs including the carboxyl terminus of DP demonstrated an accumulation of mutant protein in perinuclear aggregates within which IF subunits were sequestered. These results suggest a role for the DP carboxyl terminus in the attachment of IF to the desmosome in either a direct or indirect manner.

Desmoplakin expression and distribution in cultured rat bladder epithelial cells of varying tumorigenic potential.

The expression and distribution of the desmosomal plaque proteins, desmoplakins (DPs) I and II, were studied in nontumorigenic (RBE-8) and a series of tumorigenic (AY34, R-4909, SS-24B, RBTCC-8, and 804G) rat bladder epithelial cell lines. These cell lines ranged from slow-growing papillary transitional cells (AY34) to rapidly metastatic carcinoma cells (RBTCC-8). DPs I and II were shown by immunoblotting and Northern analysis to be present in nontumorigenic RBE-8 cells as well as in all of the tumorigenic cell lines, albeit in differing amounts. Immunofluorescence microscopy revealed striking differences in DP distribution, corresponding in general with increases in tumorigenic potential. Whereas DPs of normal RBE-8 cells and less tumorigenic AY34 cells were localized predominantly at cell interfaces, the more tumorigenic lines exhibited a high proportion of DP in the form of cytoplasmic dots, a distribution reminiscent of that seen in epithelial cells maintained in low levels of extracellular calcium. In 804G cells, which represented the most extreme example of this phenomenon, the majority of DPs were organized as cytoplasmic dots. Electron microscopy revealed intermediate filament (IF)-associated spots in the cytoplasm as well as an elaborate array of IF-associated plaques at the cell-substratum interface. The IF-associated spots in the cytoplasm reacted with anti-DP antibody in immunogold labeling experiments while those at the cell-substratum did not react. In more dense cultures of 804G cells, certain cells stratified and expressed increased amounts of DP followed by the induction of new keratins including those of the skin type. Decreasing extracellular calcium resulted in a rearrangement of DP in each cell line; staining at cell-cell interfaces disappeared and was replaced with a pattern of cytoplasmic dots. These results demonstrate a possible relationship between desmosome assembly and/or maintenance and tumorigenic potential.

Characterization of specific thyroid hormone receptors in bone.

Thyroid hormones stimulate bone turnover in vivo and increase Ca release from bone in vitro. To investigate further the effects of thyroid hormones in bone, we have characterized specific nuclear receptors for [125I]tri-iodothyronine (T3) in neonatal mouse calvaria. Maximal specific binding of [125I]T3 to isolated nuclei occurred within 60 min at 22 degrees C. [125I]T3 binding was completely and rapidly displaced by the addition of 10(-6) M unlabeled T3; the dissociation appears to be first order with t1/2 = 36 min. The IC50 for competition by unlabeled T3 was approximately 10(-8) M. The relative affinity of thyroxine (T4) for the receptor was approximately 10 X lower than T3, consistent with its lower biological potency in most target tissues for thyroid hormones. Only weak competition was observed with diiodotyrosine at concentrations up to 10(-4) M. We have previously shown that the cardiotonic agent milrinone stimulates bone resorption in vitro with characteristics similar to those of T4. Structural homology between milrinone and T4 was recently reported. Milrinone, like diiodotyrosine, was only a weak competitor for binding at concentrations up to 10(-4) M. Milrinone inhibited collagen synthesis in the calvaria. The results suggest that the effects of milrinone on bone turnover in calvaria in vitro are probably not mediated through a thyroid hormone receptor.

Cardiotonic agent milrinone stimulates resorption in rodent bone organ culture.

The cardiotonic agent amrinone inhibits bone resorption in vitro. Milrinone, an amrinone analog, is a more potent cardiotonic agent with lower toxicity. In contrast to amrinone, milrinone stimulated resorption in cultures of neonatal mouse calvaria and fetal rat limb bones. Threshold doses were 0.1 microM in calvaria and 0.1 mM in limb bones; maximal stimulation occurred in calvaria at 0.1 mM. Maximal responses to milrinone and parathyroid hormone were comparable. Milrinone concentrations below 0.1 mM did not affect calvarial cyclic AMP. 0.5 microM indomethacin inhibited milrinone effects in calvaria but usually not in limb bones. 3 nM calcitonin inhibited milrinone-stimulated resorption and there was no escape from this inhibition. Structural homology between milrinone and thyroxine has been reported. We find similarities between milrinone and thyroxine actions on bone, because prostaglandin production was crucial for the effects of both agents in calvaria but not in limb bones, and neither agent exhibited escape from calcitonin inhibition.