Introducing a null mutation in the mouse K6alpha and K6beta genes reveals their essential structural role in the oral mucosa

Mammalian genomes feature multiple genes encoding highly related keratin 6 (K6) isoforms. These type II keratins show a complex regulation with constitutive and inducible components in several stratified epithelia, including the oral mucosa and skin. Two functional genes, K6alpha and K6beta, exist in a head-to-tail tandem array in mouse genomes. We inactivated these two genes simultaneously via targeting and homologous recombination. K6 null mice are viable and initially indistinguishable from their littermates. Starting at two to three days after birth, they show a growth delay associated with reduced milk intake and the presence of white plaques in the posterior region of dorsal tongue and upper palate. These regions are subjected to greater mechanical stress during suckling. Morphological analyses implicate the filiform papillae as being particularly sensitive to trauma in K6alpha/K6beta null mice, and establish the complete absence of keratin filaments in their anterior compartment. All null mice die about a week after birth. These studies demonstrate an essential structural role for K6 isoforms in the oral mucosa, and implicate filiform papillae as being the major stress bearing structures in dorsal tongue epithelium.     

Loss of keratin 6 (K6) proteins reveals a function for intermediate filaments during wound repair

The ability to heal wounds is vital to all organisms. In mammalian tissues, alterations in intermediate filament (IF) gene expression represent an early reaction of cells surviving injury. We investigated the role of keratin IFs during the epithelialization of skin wounds using a keratin 6alpha and 6beta (K6alpha/K6beta)-null mouse model. In skin explant culture, null keratinocytes exhibit an enhanced epithelialization potential due to increased migration. The extent of the phenotype is strain dependent, and is accompanied by alterations in keratin IF and F-actin organization. However, in wounded skin in vivo, null keratinocytes rupture as they attempt to migrate under the blood clot. Fragility of the K6alpha/K6beta-null epidermis is confirmed when applying trauma to chemically treated skin. We propose that the alterations in IF gene expression after tissue injury foster a compromise between the need to display the cellular pliability necessary for timely migration and the requirement for resilience sufficient to withstand the rigors of a wound site.     

Deficiency of reproductive tract alpha(1,2)fucosylated glycans and normal fertility in mice with targeted deletions of the FUT1 or FUT2 alpha(1,2)fucosyltransferase locus.

The fucose alpha(1-->2) galactose beta structure is expressed by uterine epithelial cells in the mouse and has been implicated in blastocyst adhesion events thought to be required for murine implantation. Fucalpha(1-->2)Galbeta moieties and cognate fucosyltransferases are also expressed by epithelial cells of the male reproductive tract and have been implicated in sperm maturation events that may contribute to fertilization. To determine directly if Fucalpha(1-->2)Galbeta moieties are required for fertility, we have generated strains of mice that are deficient in genes encoding FUT1 and FUT2, a pair of GDP-L-fucose:beta(1-->4)-D-galactosyl-R 2-alpha-L-fucosyltransferase enzymes (EC 2.4.1.69) responsible for Fucalpha(1-->2)Galbeta synthesis and expression. FUT1 null mice and FUT2 null mice develop normally and exhibit no gross phenotypic abnormalities. The Fucalpha(1-->2)Galbeta epitope is absent from the uterine epithelia of FUT2 null mice and from the epithelia of the epididymis of FUT1 null mice. Fully normal fertility is observed in FUT1 null intercrosses and in FUT2 null intercrosses. These observations indicate that Fucalpha(1-->2)Galbeta moieties are not essential to blastocyst-uterine epithelial cell interactions required for implantation and are not required for sperm maturation events that permit fertilization and that neither the FUT loci nor their cognate fucosylated glycans are essential to normal development.     

Targeted disruption of the meprin beta gene in mice leads to underrepresentation of knockout mice and changes in renal gene expression profiles

Meprins are multidomain zinc metalloproteases that are highly expressed in mammalian kidney and intestinal brush border membranes and in leukocytes and certain cancer cells. Mature meprins are oligomers of evolutionarily related, separately encoded alpha and/or beta subunits. Homooligomers of meprin alpha are secreted; oligomers containing meprin beta are plasma membrane associated. Meprin substrates include bioactive peptides and extracellular matrix proteins. Meprins have been implicated in cancer and intestinal inflammation. Additionally, meprin beta is a candidate gene for diabetic nephropathy. To elucidate in vivo functions of these metalloproteases, meprin beta null mice were generated by targeted disruption of the meprin beta gene on mouse chromosome 18q12. Analyses of meprin beta knockout mice indicated that (i) 50% fewer null mice are born than the Mendelian distribution predicts, (ii) null mice that survive develop normally and are viable and fertile, (iii) meprin beta knockout mice lack membrane-associated meprin alpha in kidney and intestine, and (iv) null mice have changes in renal gene expression profiles compared to wild-type mice as assessed by microarray analyses. Thus, disruption of the meprin beta allele in mice affects embryonic viability, birth weight, renal gene expression profiles, and the distribution of meprin alpha in kidney and intestine.     

Inactivation of the integrin beta 6 subunit gene reveals a role of epithelial integrins in regulating inflammation in the lung and skin

The integrin alpha v beta 6 is only expressed in epithelial cells. In healthy adult epithelia, this receptor is barely detectable, but expression is rapidly induced following epithelial injury. Mice homozygous for a null mutation in the gene encoding the beta 6 subunit had juvenile baldness associated with infiltration of macrophages into the skin, and accumulated activated lymphocytes around conducting airways in the lungs. Beta 6-/- mice also demonstrated airway hyperresponsiveness to acetylcholine, a hallmark feature of asthma. These results suggest that the epithelial integrin alpha v beta 6 participates in the modulation of epithelial inflammation. Genetic or acquired alterations in this integrin could thus contribute to the development of inflammatory diseases of epithelial organs, such as the lungs and skin.     

Exploiting the keratin 17 gene promoter to visualize live cells in epithelial appendages of mice

Keratin genes afford, given their large number (>50) and differential regulation, a unique opportunity to study the mechanisms underlying specification and differentiation in epithelia of higher metazoans. Moreover, the small size and regulation in cis of many keratin genes enable the use of their regulatory sequence to achieve targeted gene expression in mice. Here we show that 2 kilobases of 5' upstream region from the mouse keratin 17 gene (mK17) confers expression of green fluorescent protein (GFP) in major epithelial appendages of transgenic mice. Like that of mK17, onset of [mK17 5']-GFP reporter expression coincides with the appearance of ectoderm-derived epithelial appendages during embryonic development. In adult mice, [mK17 5']-GFP is appropriately regulated within hair, nail, glands, and oral papilla. Tracking of GFP fluorescence allows for the visualization of growth cycle-related changes in hair follicles, and the defects engendered by the hairless mutation, in live skin tissue. Deletion of an internal 48-bp interval, which encompasses a Gli-responsive element, from this promoter results in loss of GFP fluorescence in most appendages in vivo, suggesting that sonic hedgehog participates in K17 regulation. The compact mK17 gene promoter provides a novel tool for appendage-preferred gene expression and manipulation in transgenic mice.     

Inhibition of integrin-mediated crosstalk with epidermal growth factor receptor/Erk or Src signaling pathways in autophagic prostate epithelial cells induces caspase-independent death

In vivo in the prostate gland, basal epithelial cells adhere to laminin 5 (LM5) via alpha3beta1 and alpha6beta4 integrins. When placed in culture primary prostate basal epithelial cells secrete and adhere to their own LM5-rich matrix. Adhesion to LM5 is required for cell survival that is dependent on integrin-mediated, ligand-independent activation of the epidermal growth factor receptor (EGFR) and the cytoplasmic tyrosine kinase Src, but not PI-3K. Integrin-mediated adhesion via alpha3beta1, but not alpha6beta4 integrin, supports cell survival through EGFR by signaling downstream to Erk. PC3 cells, which do not activate EGFR or Erk on LM5-rich matrices, are not dependent on this pathway for survival. PC3 cells are dependent on PI-3K for survival and undergo caspase-dependent death when PI-3K is inhibited. The death induced by inhibition of EGFR or Src in normal primary prostate cells is not mediated through or dependent on caspase activation, but depends on the induction of reactive oxygen species. In addition the presence of an autophagic pathway, maintained by adhesion to matrix through alpha3beta1 and alpha6beta4, prevents the induction of caspases when EGFR or Src is inhibited. Suppression of autophagy is sufficient to induce caspase activation and apoptosis in LM5-adherent primary prostate epithelial cells.     

The alpha v beta 6 integrin promotes proliferation of colon carcinoma cells through a unique region of the beta 6 cytoplasmic domain

Cell-matrix interactions are assumed to be important in regulating differentiation and tumor cell growth; however, the precise roles of individual matrix receptors in producing cellular responses are still unclear. We have previously described the alpha v beta 6 integrin, an epithelial cell fibronectin receptor expressed in many carcinoma cell lines. Here we show that heterologous expression of alpha v beta 6 in a human colon carcinoma cell line (SW480) enhances the proliferative capacity of these cells, both in vitro and in vivo in nude mice. This property of alpha v beta 6 correlates with the presence of an 11-amino acid region at the COOH terminus of the beta 6 cytoplasmic domain. This 11-amino acid sequence is required for the growth stimulatory effect, but not for other functions of the beta 6 cytoplasmic domain, such as promoting cell adhesion and focal contact localization.     

Respiratory distress and neonatal lethality in mice lacking Golgi alpha1,2-mannosidase IB involved in N-glycan maturation

There are three mammalian Golgi alpha1,2-mannosidases, encoded by different genes, that form Man5GlcNAc2 from Man(8-9)GlcNAc2 for the biosynthesis of hybrid and complex N-glycans. Northern blot analysis and in situ hybridization indicate that the three paralogs display distinct developmental and tissue-specific expression. The physiological role of Golgi alpha1,2-mannosidase IB was investigated by targeted gene ablation. The null mice have normal gross appearance at birth, but they display respiratory distress and die within a few hours. Histology of fetal lungs the day before birth indicate some delay in development, whereas neonatal lungs show extensive pulmonary hemorrhage in the alveolar region. No significant histopathological changes occur in other tissues. No remarkable ultrastructural differences are detected between wild type and null lungs. The membranes of a subset of bronchiolar epithelial cells are stained with lectins from Phaseolus vulgaris (leukoagglutinin and erythroagglutinin) and Datura stramonium in wild type lungs, but this staining disappears in lungs from null mice. Mass spectrometry of N-glycans from different tissues shows no significant changes in global N-glycans of null mice. Therefore, only a few glycoproteins required for normal lung function depend on alpha1,2-mannosidase IB for maturation. There are no apparent differences in the expression of several lung epithelial cell and endothelial cell markers between null and wild type mice. The alpha1,2-mannosidase IB null phenotype differs from phenotypes caused by ablation of other enzymes in N-glycan biosynthesis and from other mouse gene disruptions that affect pulmonary development and function.     

None of the integrins known to be present on the mouse egg or to be ADAM receptors are essential for sperm-egg binding and fusion

Antibody inhibition and alpha6beta1 ligand binding experiments indicate that the egg integrin alpha6beta1 functions as a receptor for sperm during gamete fusion; yet, eggs null for the alpha6 integrin exhibit normal fertilization. Alternative integrins may be involved in sperm-egg binding and fusion and could compensate for the absence of alpha6beta1. Various beta1 integrins and alphav integrins are present on mouse eggs. Some of these integrins are also reported to be receptors for ADAMs, which are expressed on sperm. Using alpha3 integrin null eggs, we found that the alpha3beta1 integrin was not essential for sperm-egg binding and fusion. Oocyte-specific, beta1 integrin conditional knockout mice allowed us to obtain mature eggs lacking all beta1 integrins. We found that the beta1 integrin null eggs were fully functional in fertilization both in vivo and in vitro. Furthermore, neither anti-mouse beta3 integrin function-blocking monoclonal antibody (mAb) nor alphav integrin function-blocking mAb inhibited sperm binding to or fusion with beta1 integrin null eggs. Thus, function of beta3 or alphav integrins does not seem to be involved in compensating for the absence of beta1 integrins. These results indicate that none of the integrins known to be present on mouse eggs or to be ADAM receptors are essential for sperm-egg binding/fusion, and thus, egg integrins may not play the role in gamete fusion previously attributed to them.     

Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells.

Laminin 5 regulates anchorage and motility of epithelial cells through integrins alpha6beta4 and alpha3beta1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the alpha3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all alpha3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin alpha6beta4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin alpha3beta1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin alpha3beta1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin alpha6beta4, suggesting that signaling through beta1 or beta4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium.     

Ectopic synthesis of epidermal cytokeratins in pancreatic islet cells of transgenic mice interferes with cytoskeletal order and insulin production

The members of the multigene family of intermediate filament (IF) proteins are expressed in various combinations and amounts that are specific for a given pathway or state of differentiation. Previous experiments in which the cell type-specific IF cytoskeleton was altered by introducing foreign IF proteins into cultured cells or certain tissues of transgenic animals have shown a remarkable tolerance, without detectable interference with cell functions. To examine the importance of the cell type-specific cytokeratin (CK) IF pattern, we have studied the ectopic expression of CK genes in different epithelia of transgenic mice. Here we report changes observed in the beta cells of pancreatic islets expressing the genes for human epidermal CKs 1 and/or 10 brought under control of the rat insulin promoter. Both genes were efficiently expressed, resulting in the appearance of numerous and massive bundles of aggregated IFs, resembling those of epidermal keratinocytes. While the synthesis of epidermal CK 10 was readily accommodated and compatible with cell function, mice expressing CK 1 in their beta cells, alone or in combination with CK 10, developed a special form of diabetes characterized by a drastic reduction of insulin-secretory vesicles and of insulin-and CK 1-producing cells. In many CK 1-producing cells, accumulations of fibrous or granular material containing CK 1 were also seen in the nucleus. This demonstration of functional importance of the specific CK-complement in an epithelial cell indicates a contribution of cell type-specific factors to cytoplasmic IF compartmentalization and that the specific CK complement can be crucial for functions and longevity of a given kind of epithelium.     

Heterologous expression of a mammalian epithelial sodium channel in yeast

The alpha and beta subunits of the amiloride-sensitive rat epithelial sodium channel (alpha beta ENaC) were expressed in the yeast Saccharomyces cerevisiae. We used a combination of yeast strains, including a mutant in the secretory pathway (sec6), and Western blotting techniques, to show that alpha beta ENaC was synthesized and targeted through the secretory system to the plasma membrane. Yeasts expressing alpha beta ENaC were more sensitive to salt than the parent strain. In addition, amiloride, a specific blocker of ENaC, was found to suppress salt sensitivity in the yeast strain expressing alpha beta ENaC.     

The homeobox gene Hhex is essential for proper hepatoblast differentiation and bile duct morphogenesis

Hhex is required for early development of the liver. A null mutation of Hhex results in a failure to form the liver bud and embryonic lethality. Therefore, Hhex null mice are not informative as to whether this gene is required during later stages of hepatobiliary morphogenesis. To address this question, we derived Hhex conditional null mice using the Cre-loxP system and two different Cre transgenics (Foxa3-Cre and Alfp-Cre). Deletion of Hhex in the hepatic diverticulum (Foxa3-Cre;Hhex(d2,3/-)) led to embryonic lethality and resulted in a small and cystic liver with loss of Hnf4alpha and Hnf6 expression in early hepatoblasts. In addition, the gall bladder was absent and the extrahepatic bile duct could not be identified. Loss of Hhex in the embryonic liver (Alfp-Cre;Hhex(d2,3/-)) caused irregular development of intrahepatic bile ducts and an absence of Hnf1beta in many (cystic) biliary epithelial cells, which resulted in a slow, progressive form of polycystic liver disease in adult mice. Thus, we have shown that Hhex is required during multiple stages of hepatobiliary development. The altered expression of Hnf4alpha, Hnf6 and Hnf1beta in Hhex conditional null mice suggests that Hhex is an essential component of the genetic networks regulating hepatoblast differentiation and intrahepatic bile duct morphogenesis.     

Characterization of the translational start site for IF2 beta, a short form of Escherichia coli initiation factor IF2

The gene for initiation factor IF2, infB, represents one of the few examples in Escherichia coli of genes encoding two protein products in vivo. In a previous work, our group showed that both forms of IF2 (alpha and beta) are closely related and may arise from two independent translational events on infB mRNA. Unambiguous mapping and rigorous determination of the nature of the initiation triplet for IF2 beta, the smaller form of IF2, is critical for future mutagenesis of this codon, required for investigating the biological importance of both IF2 alpha and IF2 beta. Three types of experiments were carried out. First, a 77-bp deletion was created at the beginning of the structural gene leading to premature termination of IF2 alpha synthesis. Under these conditions, IF2 beta is still formed. Second, various Bal31 digests of infB containing the 77-bp deletion were fused to lacZ. Any synthesis of a fused protein with beta-galactosidase activity should reflect the occurrence of an initiation event on the messenger corresponding to this DNA segment. It was consequently possible to locate the IF2 beta initiation site within an 18-base region containing an in-phase GUG codon. Third, to avoid any artefactual reinitiation event possibly occurring under our experimental conditions, we fused to lacZ an infB fragment devoid of IF2 alpha start sequences but containing genetic information for this 18-base region. A hybrid protein with beta-galactosidase activity was synthesized. Moreover, its NH2-terminal amino acid sequence coincided with that of IF2 beta, demonstrating that GUG, located 471 bases downstream from the IF2 alpha external start codon, is the internal start codon for the shorter form of IF2.     

Loss of assembly of the main basement membrane collagen, type IV, but not fibril-forming collagens and embryonic death in collagen prolyl 4-hydroxylase I null mice

Collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of the 4-hydroxyproline residues that are essential for the generation of triple helical collagen molecules. The vertebrate C-P4Hs I, II, and III are [alpha(I)]2beta2, [alpha(II)]2beta2, and [alpha(III)]2beta2 tetramers with identical beta subunits. We generated mice with targeted inactivation of the P4ha1 gene encoding the catalytic alpha subunit of C-P4H I to analyze its specific functions. The null mice died after E10.5, showing an overall developmental delay and a dilated endoplasmic reticulum in their cells. The capillary walls were frequently ruptured, but the capillary density remained unchanged. The C-P4H activity level in the null embryos and fibroblasts cultured from them was 20% of that in the wild type, being evidently due to the other two isoenzymes. Collagen IV immunofluorescence was almost absent in the basement membranes of the null embryos, and electron microscopy revealed disrupted basement membranes, while immunoelectron microscopy showed a lack of collagen IV in them. The amount of soluble collagen IV was increased in the null embryos and cultured null fibroblasts, indicating a lack of assembly of collagen IV molecules into insoluble structures, probably due to their underhydroxylation and hence abnormal conformation. In contrast, the null embryos had collagen I and III fibrils with a typical cross-striation pattern but slightly increased diameters, and the null fibroblasts secreted fibril-forming collagens, although less efficiently than wild-type cells. The primary cause of death of the null embryos was thus most likely an abnormal assembly of collagen IV.