Purine nucleoside phosphorylase deficiency: a new case report and identification of two novel mutations (Gly156A1a and Val217Ile), only one of which (Gly156A1a) is deleterious.

Purine nucleoside phosphorylase (PNP) deficiency results in an autosomal recessive immunodeficiency disease characterized by initial involvement of cellular immunity and neurological manifestations with subsequent abnormalities of humoral immunity. The initial presentation and clinical course has varied widely in the relatively few published cases. The molecular basis has been reported in only 10 patients, precluding evaluation of phenotype-genotype relationships. We now report clinical, immunologic, and molecular findings in a new case of relatively early onset that emphasizes hypotonia and developmental delay as early manifestations. The patient carried two novel missense mutations (Gly56A1a and Val217Ile) on the same allele in apparent homozygosity. Expression of each of the mutant enzymes in vitro demonstrated that the Gly156A1a mutation abolished enzyme activity while the Val217Ile mutation was without obvious effect and is therefore a normal variant. Such "normal" polymorphisms might be associated with a variable response to the immunosuppressive PNP inhibitors currently in clinical trials.

Wound healing is accelerated by agonists of adenosine A2 (G alpha s-linked) receptors.

The complete healing of wounds is the final step in a highly regulated response to injury. Although many of the molecular mediators and cellular events of healing are known, their manipulation for the enhancement and acceleration of wound closure has not proven practical as yet. We and others have established that adenosine is a potent regulator of the inflammatory response, which is a component of wound healing. We now report that ligation of the G alpha s-linked adenosine receptors on the cells of an artificial wound dramatically alters the kinetics of wound closure. Excisional wound closure in normal, healthy mice was significantly accelerated by topical application of the specific A2A receptor agonist CGS-21680 (50% closure by day 2 in A2 receptor antagonists. In rats rendered diabetic (streptozotocin-induced diabetes mellitus) wound healing was impaired as compared to nondiabetic rats; CGS-21680 significantly increased the rate of wound healing in both nondiabetic and diabetic rats. Indeed, the rate of wound healing in the CGS-21680-treated diabetic rats was greater than or equal to that observed in untreated normal rats. These results appear to constitute the first evidence that a small molecule, such as an adenosine receptor agonist, accelerates wound healing in both normal animals and in animals with impaired wound healing.

Two newly identified mutations (Thr233Ile and Leu152Met) in partially adenosine deaminase-deficient (ADA-) individuals that result in differing biochemical and metabolic phenotypes.

Deficiency of adenosine deaminase (ADA-) results in autosomal recessive immunodeficiency disease of varying severity. Partial ADA- [ADA deficiency in erythrocytes (RBCs) but substantial ADA in non-RBCs] has also been identified, primarily by population screening of healthy adults in Africa and newborns in New York State. Normal immune function and/or minimal elevations of toxic metabolites in childhood suggested that partial ADA deficiency was benign and therefore that six mutations identified in partially ADA-deficient newborns and expressing 8-80% of normal ADA in non-RBCs were not pathogenic. However, the lowest activity mutation (Arg211Cys) has now been reported in patients with adult-onset immunodeficiency. We have now molecularly and biochemically studied two additional individuals whom we found to represent opposite ends of the spectrum of partial ADA deficiency as to biochemical abnormalities and age of ascertainment. Homozygosity for a newly identified Leu152Met mutation expressing considerably less activity than the pathogenic Arg211Cys mutation was found in a currently healthy 10-year-old Afghanistani child (ascertained at birth). He had the highest accumulation of the metabolite dATP among 13 partially ADA-deficient patients studied, but considerably lower than in those with immunodeficiency. Homozygosity for a newly identified Thr233Ile mutation expressing somewhat greater ADA activity than Arg211Cys was found in a healthy young adult Kung individual, associated with very low metabolite concentrations. Biochemical findings and a family history suggestive of immunodeficiency in prior offspring support the idea that the Leu152Met mutation could result in disease in homozygous individuals challenged by severe environmental insult or in heterozygosity with a null mutation. The pathogenicity of the Thr233Ile mutation, as well as a previously described Ala215Thr mutation with relatively lower activity is less likely but will only be determined by long-term observation of individuals carrying these mutations. Although, in contrast to other partial mutations, neither of these two mutations are at CpG hot spots, the frequency of CpG mutations remains high for partial mutations but is also similarly high in ADA- immunodeficient patients (5/8 vs 12/21).

Spontaneous in vivo reversion to normal of an inherited mutation in a patient with adenosine deaminase deficiency.

Somatic mosaicism in genetic disease generally results from a de novo deleterious mutation during embryogenesis. We now describe a somatic mosaicism due to the unusual mechanism of in vivo reversion to normal of an inherited mutation. The propositus was an adenosine deaminase-deficient (ADA-) child with progressive clinical improvement and unexpectedly mild biochemical and immunologic abnormalities. Mosaicism due to reversion was evidenced by absence of a maternally transmitted deleterious mutation in 13/15 authenticated B cell lines and in 17% of single alleles cloned from blood DNA, despite retention of a maternal 'private' ADA polymorphism linked to the mutation. Establishment of significant somatic mosaicism following reversion to normal could modify any disorder in which revertant cells have a selective advantage.

TGF beta promotes the basal phenotype of epidermal keratinocytes: transcriptional induction of K#5 and K#14 keratin genes.

TGFbeta is an important regulator of epidermal keratinocyte function because it suppresses cell proliferation, while it induces synthesis of extracellular matrix proteins and their cells surface receptors. To examine whether TGFbeta affects synthesis of intracellular proteins as well, specifically the transcription of keratin genes, we transfected a series of DNA constructs that contain keratin gene promoters into human epidermal keratinocytes. The transfected cells were grown in the presence and absence of TGFbeta. We found that TGFbeta specifically induces transcription controlled by the promoters of K#5 and K#14 keratin genes, markers of basal cells. No other keratin gene promoters were induced. The effect of TGFbeta is concentration-dependent, can be demonstrated in HeLa cells, does not depend on keratinocyte growth conditions and can be elicited by both TGFbeta1 and TGFbeta2. We conclude that TGFbeta promotes the basal cell phenotype in stratified epithelia such as the epidermis.

Disease-activated transcription factor: allergic reactions in human skin cause nuclear translocation of STAT-91 and induce synthesis of keratin K17.

Epidermal keratinocytes have important immunologic functions, which is apparent during wound healing, in psoriasis, and in allergic and inflammatory reactions. In these processes, keratinocytes not only produce cytokines and growth factors that attract and affect lymphocytes but also respond to the polypeptide factors produced by the lymphocytes. Gamma interferon (IFN-gamma) is one such signaling polypeptide. Its primary molecular effect is activation of specific transcription factors that regulate gene expression in target cells. In this work, we present a molecular mechanism of lymphocyte-keratinocyte signaling in the epidermis. We have induced cutaneous delayed-type hypersensitivity reactions that are associated with an accumulation of lymphocytes. These resulted in activation and nuclear translocation of STAT-91, the IFN-gamma-activated transcription factor, in keratinocytes in vivo and subsequent induction of transcription of keratin K17. Within the promoter of the K17 keratin gene, we have identified and characterized a site that confers the responsiveness to IFN-gamma and that binds the transcription factor STAT-91. Other keratin gene promoters tested were not induced by IFN-gamma. These results characterize at the molecular level a signaling pathway produced by the infiltration of lymphocytes in skin and resulting in the specific alteration of gene expression in keratinocytes.

Epidermal growth factor and transforming growth factor alpha specifically induce the activation- and hyperproliferation-associated keratins 6 and 16.

Epidermal injury results in activation of keratinocytes which produce and respond to growth factors and cytokines and become migratory. Activated keratinocytes express a specific pair of keratin proteins, K6 and K16, distinct from the keratins in the healthy epidermis. Keratinocytes can be activated, for example, by binding of the appropriate ligands to the epidermal growth factor receptor (EGFR). We have analyzed the effects of EGFR activation on keratin gene transcription by transfecting DNAs containing keratin promoters linked to a reporter gene into primary cultures of human epidermal keratinocytes in the presence or absence of EGF or transforming growth factor alpha (TGF alpha), two growth factors that activate EGFR. The activation of EGFR had no effect on the promoters of simple epithelial, basal-layer-specific, or differentiation-specific keratins. In contrast, the expression of K6 and K16 was strongly and specifically induced. A 20-bp DNA segment of the K16 gene promoter conveyed the EGF regulation, functioned in a heterologous construct, and therefore constituted an EGF-responsive element. A nuclear protein specifically bound to this element and to the analogous sequence of the K6 promoter. Thus, EGF specifically induces K6 and K16, markers of activated keratinocytes, via nuclear proteins that bind to EGF-responsive elements in the promoters of these keratin genes.

Vitamin D3, its receptor and regulation of epidermal keratin gene expression.

Among extrinsic modulators of keratinization are certain hormones and vitamins, which makes them potentially important pharmacological tools for treatment of keratinization disorders. Vitamin D3 and vitamin A, and their metabolites, promote and inhibit keratinization, respectively. We have shown that retinoic acid, via its nuclear receptor, directly suppresses the expression of the keratin genes which are markers of keratinocyte differentiation. Here we present evidence that 1,25(OH)2 vitamin D3 and its nuclear receptor do not directly regulate keratin gene expression. Co-transfection of a vector expressing the nuclear receptor for vitamin D3 with responder DNA constructs containing keratin gene promoters had no effect on the level of activity of keratin gene promoters either in the presence or in the absence of vitamin D3. We conclude that vitamin D3, unlike retinoic acid, modifies keratin synthesis indirectly, by changing the differentiation phenotype of the keratinocyte.

Comparison of methods for transfection of human epidermal keratinocytes.

Several methods for DNA-mediated cell transfection were tested to determine the optimal conditions for transfection of human epidermal keratinocytes. The following methods were compared: electroporation, lipofection, Ca3(PO4)2 co-precipitation, DEAE-dextran, and polybrene-mediated transfection. The transfected DNA included human keratinocyte-specific promoter for keratin K14 as well as SV40 and RSV viral promoters. Enzyme assays and in situ staining were used to evaluate both quantitative and qualitative aspects of transfection, and both subconfluent and post-confluent, stratifying keratinocytes were examined. Lipofection, Ca3(PO4)2 co-precipitation, and polybrene methods transfect very efficiently, but lipofection is expensive and Ca++ in the co-precipitation procedure induces keratinocytes to differentiate. We have found that polybrene-mediated transfection followed by a 27% DMSO shock is optimal for introducing DNA into human epidermal keratinocytes.

Functional comparison of the upstream regulatory DNA sequences of four human epidermal keratin genes.

The promoters of epidermal keratin genes, K5, K6, and K10 were cloned and their functions compared with that of the previously described promoter of the K14 keratin gene in non-epithelial and transformed epithelial cell lines, as well as in primary cultures of cells derived from simple and stratified epithelia. The four promoters were functional only in epithelial cells. Although the promoter for the basal cell-specific, acidic-type K14 gene was active in all epithelial cells tested, its basic-type partner, K5, and the promoter for the hyper-proliferation-associated K6 were active only in primary cultures of stratified epithelia. The promoter for the epidermal differentiation-specific K10 keratin gene was active at a low level in primary cultures of stratified epithelial cells on non-epidermal origin. Thus, the K14 gene promoter is functional in all epithelial cells, but the upstream regions of the K5 and K6 keratin genes restrict their expression to stratified epithelia, whereas the epidermal determinants of the K10 gene are not in the proximal upstream sequences.

[A novel monoclonal antibody against human keratins].

In this paper, we reported a novel monoclonal antibody against human keratins, R 6-2-14. The antigen used for immunization was derived from human callus, keratins in which traditionally are classified as "Soft" keratins. However, when we studied the tissue specificity of this antibody, it was found that it only reacted strongly with "Hard" keratins of various mammalian species, but no detectable cross-reactivity with any of the "Soft" keratins. This antibody may provide a useful tool for the study of hair regeneration, nail regeneration, corn pathology and differentiation of mammalian epidermal derivatives.

Nuclear receptors for retinoic acid and thyroid hormone regulate transcription of keratin genes.

In the epidermis, retinoids regulate the expression of keratins, the intermediate filament proteins of epithelial cells. We have cloned the 5' regulatory regions of four human epidermal keratin genes, K#5, K#6, K#10, and K#14, and engineered constructs in which these regions drive the expression of the CAT reporter gene. By co-transfecting the constructs into epithelial cells along with the vectors expressing nuclear receptors for retinoic acid (RA) and thyroid hormone, we have demonstrated that the receptors can suppress the promoters of keratin genes. The suppression is ligand dependent; it is evident both in established cell lines and in primary cultures of epithelial cells. The three RA receptors have similar effects on keratin gene transcription. Our data indicate that the nuclear receptors for RA and thyroid hormone regulate keratin synthesis by binding to negative recognition elements in the upstream DNA sequences of the keratin genes. RA thus has a twofold effect on epidermal keratin expression: qualitatively, it regulates the regulators that effect the switch from basal cell-specific keratins to differentiation-specific ones; and quantitatively, it determines the level of keratin synthesis within the cell by direct interaction of its receptors with the keratin gene promoters.

Epithelial-specific keratin gene expression: identification of a 300 base-pair controlling segment.

To elucidate the elements required for regulation of keratin expression in epidermis, we have linked a short, 300 base pair segment, corresponding to the promoter region of a human K#14 gene, to the chloramphenicol-acetyl-transferase gene. This construct was introduced into various mammalian cell lines and primary cultures via Ca3(PO4)2 precipitation. The 300 base pair segment from the keratin gene promoter region was active in all epithelial cells studied including transformed, simple epithelial cells such as HeLa and ME-180, cell lines derived from stratified epithelium, such as SCC-12, as well as primary cultures of epithelial cells. The construct was inactive in all non-epithelial cells tested including fibroblasts and melanocytes. The segment does not function as a silencer in nonepithelial cells but it can function as an enhancer in epithelial cells. Using the polymerase chain reaction we have constructed a series of deletions of the promoter and have localized an essential function within a 40 bp sequence. We conclude that we have identified the keratin gene promoter that is sufficient to confer epithelial-specific expression.

Embryonic expression of the human 40-kD keratin: evidence from a processed pseudogene sequence.

Analysis of the cytoskeletal components of early murine embryos has detected expression of two keratin proteins, K#8 and K#18, at the 4-8-cell stage. Comparable data for human embryos do not exist, although several processed pseudogenes corresponding to K#8 and K#18 have been discovered in the human genome. Because only genes that are expressed in pre-germ-line and germ-line cells can give rise to processed pseudogenes, the existence of human K#8 and K#18 processed pseudogenes is prima facie evidence for expression of keratins K#8 and K#18 in the early human embryo. We have cloned and determined the complete sequence of a processed pseudogene corresponding to another acidic human keratin. Comparison of its sequence with known sequences of other mammalian keratins indicates that the pseudogene arose from a reverse transcript of a correctly initiated and terminated functional human K#19 gene. This implies expression of K#19 keratin in addition to K#8 and K#18 in the early human embryo. We have proposed previously that K#19 evolved specifically to redress unbalanced production of various basic keratins, and our current evidence, that it is expressed at an early stage of development, implies that K#19 may fulfill this same role during human embryogenesis.