Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems.

UBE2W ubiquitinates N termini of proteins rather than internal lysine residues, showing a preference for substrates with intrinsically disordered N termini. The in vivo functions of this intriguing E2, however, remain unknown. We generated Ube2w germ line KO mice that proved to be susceptible to early postnatal lethality without obvious developmental abnormalities. Although the basis of early death is uncertain, several organ systems manifest changes in Ube2w KO mice. Newborn Ube2w KO mice often show altered epidermal maturation with reduced expression of differentiation markers. Mirroring higher UBE2W expression levels in testis and thymus, Ube2w KO mice showed a disproportionate decrease in weight of these two organs (~50%), suggesting a functional role for UBE2W in the immune and male reproductive systems. Indeed, Ube2w KO mice displayed sustained neutrophilia accompanied by increased G-CSF signaling and testicular vacuolation associated with decreased fertility. Proteomic analysis of a vulnerable organ, presymptomatic testis, showed a preferential accumulation of disordered proteins in the absence of UBE2W, consistent with the view that UBE2W preferentially targets disordered polypeptides. These mice further allowed us to establish that UBE2W is ubiquitously expressed as a single isoform localized to the cytoplasm and that the absence of UBE2W does not alter cell viability in response to various stressors. Our results establish that UBE2W is an important, albeit not essential, protein for early postnatal survival and normal functioning of multiple organ systems.

Human ZMPSTE24 disease mutations: residual proteolytic activity correlates with disease severity.

The zinc metalloprotease ZMPSTE24 plays a critical role in nuclear lamin biology by cleaving the prenylated and carboxylmethylated 15-amino acid tail from the C-terminus of prelamin A to yield mature lamin A. A defect in this proteolytic event, caused by a mutation in the lamin A gene (LMNA) that eliminates the ZMPSTE24 cleavage site, underlies the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Likewise, mutations in the ZMPSTE24 gene that result in decreased enzyme function cause a spectrum of diseases that share certain features of premature aging. Twenty human ZMPSTE24 alleles have been identified that are associated with three disease categories of increasing severity: mandibuloacral dysplasia type B (MAD-B), severe progeria (atypical 'HGPS') and restrictive dermopathy (RD). To determine whether a correlation exists between decreasing ZMPSTE24 protease activity and increasing disease severity, we expressed mutant alleles of ZMPSTE24 in yeast and optimized in vivo yeast mating assays to directly compare the activity of alleles associated with each disease category. We also measured the activity of yeast crude membranes containing the ZMPSTE24 mutant proteins in vitro. We determined that, in general, the residual activity of ZMPSTE24 patient alleles correlates with disease severity. Complete loss-of-function alleles are associated with RD, whereas retention of partial, measureable activity results in MAD-B or severe progeria. Importantly, our assays can discriminate small differences in activity among the mutants, confirming that the methods presented here will be useful for characterizing any new ZMPSTE24 mutations that are discovered.

p38 Inhibition ameliorates skin and skull abnormalities in Fgfr2 Beare-Stevenson mice.

Beare-Stevenson cutis gyrata syndrome (BSS) is a human genetic disorder characterized by skin and skull abnormalities. BSS is caused by mutations in the FGF receptor 2 (FGFR2), but the molecular mechanisms that induce skin and skull abnormalities are unclear. We developed a mouse model of BSS harboring a FGFR2 Y394C mutation and identified p38 MAPK as an important signaling pathway mediating these abnormalities. Fgfr2+/Y394C mice exhibited epidermal hyperplasia and premature closure of cranial sutures (craniosynostosis) due to abnormal cell proliferation and differentiation. We found ligand-independent phosphorylation of FGFR2 and activation of p38 signaling in mutant skin and calvarial tissues. Treating Fgfr2+/Y394C mice with a p38 kinase inhibitor attenuated skin abnormalities by reversing cell proliferation and differentiation to near normal levels. This study reveals the pleiotropic effects of the FGFR2 Y394C mutation evidenced by cutis gyrata, acanthosis nigricans, and craniosynostosis and provides a useful model for investigating the molecular mechanisms of skin and skull development. The demonstration of a pathogenic role for p38 activation may lead to the development of therapeutic strategies for BSS and related conditions, such as acanthosis nigricans or craniosynostosis.

Kindlin-1 Is required for RhoGTPase-mediated lamellipodia formation in keratinocytes.

Kindlin-1 is an epithelial-specific member of the novel kindlin protein family, which are regulators of integrin functions. Mutations in the gene that encodes Kindlin-1, FERMT1 (KIND1), cause the Kindler syndrome (KS), a human disorder characterized by mucocutaneous fragility, progressive skin atrophy, ulcerative colitis, photosensitivity, and propensity to skin cancer. Our previous studies indicated that loss of kindlin-1 resulted in abnormalities associated with integrin functions, such as adhesion, proliferation, polarization, and motility of epidermal cells. Here, we disclosed novel FERMT1 mutations in KS and used them, in combination with small-interfering RNA, protein, and imaging studies, to uncover new functions for kindlin-1 in keratinocytes and to discern the molecular pathology of KS. We show that kindlin-1 forms molecular complexes with beta1 integrin, alpha-actinin, migfilin, and focal adhesion kinase and regulates cell shape and migration by controlling lamellipodia formation. Kindlin-1 governs these processes by signaling via Rho family GTPases, and it is required to maintain the pool of GTP-bound, active Rac1, RhoA and Cdc42, and the phosphorylation of their downstream effectors p21-activated kinase 1, LIM kinase, and cofilin. Loss of these kindlin-1 functions forms the biological basis for the epithelial cell fragility and atrophy in the pathology of KS.

Cardiofaciocutaneous (CFC) syndrome associated with muscular coenzyme Q10 deficiency.

The cardiofaciocutaneous (CFC) syndrome is characterized by congenital heart defect, developmental delay, peculiar facial appearance with bitemporal constriction, prominent forehead, downslanting palpebral fissures, curly sparse hair and abnormalities of the skin. CFC syndrome phenotypically overlaps with Noonan and Costello syndromes. Mutations of several genes (PTPN11, HRAS, KRAS, BRAF, MEK1 and MEK2), involved in the mitogen-activated protein kinase (MAPK) pathway, have been identified in CFC-Costello-Noonan patients. Coenzyme Q10 (CoQ10), a lipophilic molecule present in all cell membranes, functions as an electron carrier in the mitochondrial respiratory chain, where it transports electrons from complexes I and II to complex III. CoQ10 deficiency is a rare treatable mitochondrial disorder with various neurological (cerebellar ataxia, myopathy, epilepsy, mental retardation) and extraneurological (cardiomyopathy, nephropathy) signs that are responsive to CoQ10 supplementation. We report the case of a 4-year-old girl who presented a CFC syndrome, confirmed by the presence of a pathogenic R257Q BRAF gene mutation, together with a muscular CoQ10 deficiency. Her psychomotor development was severely impaired, hindered by muscular hypotonia and ataxia, both improving remarkably after CoQ10 treatment. This case suggests that there is a functional connection between the MAPK pathway and the mitochondria. This could be through the phosphorylation of a nuclear receptor essential for CoQ10 biosynthesis. Another hypothesis is that K-Ras, one of the proteins composing the MAPK pathway, might be recruited into the mitochondria to promote apoptosis. This case highlights that CoQ10 might contribute to the pathogenesis of CFC syndrome.

Mutations of the mitochondrial holocytochrome c-type synthase in X-linked dominant microphthalmia with linear skin defects syndrome.

The microphthalmia with linear skin defects syndrome (MLS, or MIDAS) is an X-linked dominant male-lethal disorder almost invariably associated with segmental monosomy of the Xp22 region. In two female patients, from two families, with MLS and a normal karyotype, we identified heterozygous de novo point mutations--a missense mutation (p.R217C) and a nonsense mutation (p.R197X)--in the HCCS gene. HCCS encodes the mitochondrial holocytochrome c-type synthase that functions as heme lyase by covalently adding the prosthetic heme group to both apocytochrome c and c(1). We investigated a third family, displaying phenotypic variability, in which the mother and two of her daughters carry an 8.6-kb submicroscopic deletion encompassing part of the HCCS gene. Functional analysis demonstrates that both mutant proteins (R217C and Delta 197-268) were unable to complement a Saccharomyces cerevisiae mutant deficient for the HCCS orthologue Cyc3p, in contrast to wild-type HCCS. Moreover, ectopically expressed HCCS wild-type and the R217C mutant protein are targeted to mitochondria in CHO-K1 cells, whereas the C-terminal-truncated Delta 197-268 mutant failed to be sorted to mitochondria. Cytochrome c, the final product of holocytochrome c-type synthase activity, is implicated in both oxidative phosphorylation (OXPHOS) and apoptosis. We hypothesize that the inability of HCCS-deficient cells to undergo cytochrome c-mediated apoptosis may push cell death toward necrosis that gives rise to severe deterioration of the affected tissues. In summary, we suggest that disturbance of both OXPHOS and the balance between apoptosis and necrosis, as well as the X-inactivation pattern, may contribute to the variable phenotype observed in patients with MLS.

Farnesylated lamins, progeroid syndromes and farnesyl transferase inhibitors.

Three mammalian nuclear lamin proteins, lamin B(1), lamin B(2) and the lamin A precursor, prelamin A, undergo canonical farnesylation and processing at CAAX motifs. In the case of prelamin A, there is an additional farnesylation-dependent endoproteolysis, which is defective in two congenital diseases: Hutchinson-Gilford progeria (HGPS) and restrictive dermopathy (RD). These two diseases arise respectively from defects in the prelamin A substrate and the enzyme (ZmpSte24) that processes it. Recent work has shed light on the roles of the lamin proteins and the enzymes involved in their farnesylation-dependent maturation. Other experimental work, including mouse model studies, have examined the possibility that farnesyl transferase inhibitors can represent effective treatment for HGPS. However, there are concerns about their use for this purpose given the potential for alternative prenylation pathways.

Scd1ab-Xyk: a new asebia allele characterized by a CCC trinucleotide insertion in exon 5 of the stearoyl-CoA desaturase 1 gene in mouse.

We describe here a spontaneous, autosomal recessive mutant mouse suffering from skin and hair defects, which arose in the outbred Kunming strain. By haplotype analysis and direct sequencing of PCR products, we show that this mutation is a new allele of the asebia locus with a naturally occurring mutation in the Scd1 gene (a CCC insertion at nucleotide position 835 in exon 5), which codes for stearoyl-CoA desaturase 1. This mutation introduces an extra proline residue at position 279 in the Scd1 protein. The mutant mice, originally designated km/km but now assigned the name Scd1ab-Xyk (hereafter abbreviated as abXyk/abXyk), have a similar gross and histological phenotype to that reported for previously characterized allelic asebia mutations (Scd1ab, Scd1abJ, Scd1ab2J, and Scd1tm1Ntam). Histological analysis showed they were also characterized by hypoplasic sebaceous glands and abnormal hair follicles. In a cross between Kunming- abXyk/abXyk and ABJ/Le-abJ/abJ mice, all the progeny showed the same phenotype, indicating that the two mutations were non-complementing and therefore allelic. Comparisons with the other four allelic mutants indicate that the Scd1ab-Xyk mutation causes the mildest change in Scd1 function. This new mouse mutant is a good model not only for the study of scarring alopecias in humans, which are characterized by hypoplasic sebaceous glands, but also for studying the structure and function of the Scd1 protein.

Loss of proteolytically processed filaggrin caused by epidermal deletion of Matriptase/MT-SP1.

Profilaggrin is a large epidermal polyprotein that is proteolytically processed during keratinocyte differentiation to release multiple filaggrin monomer units as well as a calcium-binding regulatory NH2-terminal filaggrin S-100 protein. We show that epidermal deficiency of the transmembrane serine protease Matriptase/MT-SP1 perturbs lipid matrix formation, cornified envelope morphogenesis, and stratum corneum desquamation. Surprisingly, proteomic analysis of Matriptase/MT-SP1-deficient epidermis revealed the selective loss of both proteolytically processed filaggrin monomer units and the NH2-terminal filaggrin S-100 regulatory protein. This was associated with a profound accumulation of profilaggrin and aberrant profilaggrin-processing products in the stratum corneum. The data identify keratinocyte Matriptase/MT-SP1 as an essential component of the profilaggrin-processing pathway and a key regulator of terminal epidermal differentiation.

Keratinocyte-specific Pten deficiency results in epidermal hyperplasia, accelerated hair follicle morphogenesis and tumor formation.

PTEN is a tumor suppressor gene mutated in many human cancers. We used the Cre-loxP system to generate a keratinocyte-specific null mutation of Pten in mice (k5Pten(flox/flox) mice). k5Pten(flox/flox) mice exhibit wrinkled skin because of epidermal hyperplasia and hyperkeratosis and ruffled, shaggy, and curly hair. Histological examination revealed that skin morphogenesis is accelerated in k5Pten(flox/flox) mice. Within 3 weeks of birth, 90% of k5Pten(flox/flox) mice die of malnutrition possibly caused by hyperkeratosis of the esophagus. All k5Pten(flox/flox) mice develop spontaneous tumors within 8.5 months of birth, and chemical treatment accelerates the onset of tumors. k5Pten(flox/flox) keratinocytes are hyperproliferative and resistant to apoptosis and show increased activation of the Pten downstream signaling mediators Akt/protein kinase B (PKB) and extracellular signal-regulated kinase. Pten is thus an important regulator of normal development and oncogenesis in the skin.

Matriptase/MT-SP1 is required for postnatal survival, epidermal barrier function, hair follicle development, and thymic homeostasis.

Matriptase/MT-SP1 is a novel tumor-associated type II transmembrane serine protease that is highly expressed in the epidermis, thymic stroma, and other epithelia. A null mutation was introduced into the Matriptase/MT-SP1 gene of mice to determine the role of Matriptase/MT-SP1 in epidermal development and neoplasia. Matriptase/MT-SP1-deficient mice developed to term but uniformly died within 48 h of birth. All epidermal surfaces of newborn mice were grossly abnormal with a dry, red, shiny, and wrinkled appearance. Matriptase/MT-SP1-deficiency caused striking malformations of the stratum corneum, characterized by dysmorphic and pleomorphic corneocytes and the absence of vesicular bodies in transitional layer cells. This aberrant skin development seriously compromised both inward and outward epidermal barrier function, leading to the rapid and fatal dehydration of Matriptase/MT-SP1-deficient pups. Loss of Matriptase/MT-SP1 also seriously affected hair follicle development resulting in generalized follicular hypoplasia, absence of erupted vibrissae, lack of vibrissal hair canal formation, ingrown vibrissae, and wholesale abortion of vibrissal follicles. Furthermore, Matriptase/MT-SP1-deficiency resulted in dramatically increased thymocyte apoptosis, and depletion of thymocytes. This study demonstrates that Matriptase/MT-SP1 has pleiotropic functions in the development of the epidermis, hair follicles, and cellular immune system.

The gene mutated in bare patches and striated mice encodes a novel 3beta-hydroxysteroid dehydrogenase.

X-linked dominant disorders that are exclusively lethal prenatally in hemizygous males have been described in human and mouse. None of the genes responsible has been isolated in either species. The bare patches (Bpa) and striated (Str) mouse mutations were originally identified in female offspring of X-irradiated males. Subsequently, additional independent alleles were described. We have previously mapped these X-linked dominant, male-lethal mutations to an overlapping region of 600 kb that is homologous to human Xq28 (ref. 4) and identified several candidate genes in this interval. Here we report mutations in one of these genes, Nsdhl, encoding an NAD(P)H steroid dehydrogenase-like protein, in two independent Bpa and three independent Str alleles. Quantitative analysis of sterols from tissues of affected Bpa mice support a role for Nsdhl in cholesterol biosynthesis. Our results demonstrate that Bpa and Str are allelic mutations and identify the first mammalian locus associated with an X-linked dominant, male-lethal phenotype. They also expand the spectrum of phenotypes associated with abnormalities of cholesterol metabolism.

Limb and skin abnormalities in mice lacking IKKalpha.

The gene encoding inhibitor of kappa B (IkappaB) kinase alpha (IKKalpha; also called IKK1) was disrupted by gene targeting. IKKalpha-deficient mice died perinatally. In IKKalpha-deficient fetuses, limb outgrowth was severely impaired despite unaffected skeletal development. The epidermal cells in IKKalpha-deficient fetuses were highly proliferative with dysregulated epidermal differentiation. In the basal layer, degradation of IkappaB and nuclear localization of nuclear factor kappa B (NF-kappaB) were not observed. Thus, IKKalpha is essential for NF-kappaB activation in the limb and skin during embryogenesis. In contrast, there was no impairment of NF-kappaB activation induced by either interleukin-1 or tumor necrosis factor-alpha in IKKalpha-deficient embryonic fibroblasts and thymocytes, indicating that IKKalpha is not essential for cytokine-induced activation of NF-kappaB.

Abnormal morphogenesis but intact IKK activation in mice lacking the IKKalpha subunit of IkappaB kinase.

The oligomeric IkappaB kinase (IKK) is composed of three polypeptides: IKKalpha and IKKbeta, the catalytic subunits, and IKKgamma, a regulatory subunit. IKKalpha and IKKbeta are similar in structure and thought to have similar function-phosphorylation of the IkappaB inhibitors in response to proinflammatory stimuli. Such phosphorylation leads to degradation of IkappaB and activation of nuclear factor kappaB transcription factors. The physiological function of these protein kinases was explored by analysis of IKKalpha-deficient mice. IKKalpha was not required for activation of IKK and degradation of IkappaB by proinflammatory stimuli. Instead, loss of IKKalpha interfered with multiple morphogenetic events, including limb and skeletal patterning and proliferation and differentiation of epidermal keratinocytes.

Defective stratum corneum and early neonatal death in mice lacking the gene for transglutaminase 1 (keratinocyte transglutaminase).

The stratum corneum of the skin serves as an effective barrier for maintenance of the internal milieu against the external environment. At the cell periphery of the stratum corneum is the cell envelope, a highly insoluble membranous structure composed of precursor proteins cross-linked by epsilon-(gamma-glutamyl)lysine bonds. Transglutaminase 1 (TGase 1; keratinocyte TGase), a membrane-bound isozyme of the TGase family, has been proposed to catalyze this process of assembly. Deficient cross-linking of the cell envelope in some patients with the autosomal recessive skin disorder lamellar ichthyosis (LI) and several mutations of the TGase 1 gene that have been identified in families with LI suggest the importance of this gene in production of the cell envelope. In this study, we generated mice lacking the TGase 1 gene, and we report that they have erythrodermic skin with abnormal keratinization. In their stratum corneum, degradation of nuclei and keratohyalin F-granules was incomplete and cell envelope assembly was defective. The skin barrier function of TGase 1-null mice was markedly impaired, and these mice died within 4-5 h after birth. These results clearly demonstrate that the TGase 1 gene is essential to the development and maturation of the stratum corneum and to adaptation to the environment after birth. Thus, these TGase 1 knockout mice may be a useful model for severe cases of LI.