K10 mRNA Research Articles

Coordinate changes in gene expression which mark the spinous to granular cell transition in epidermis are regulated by protein kinase C.

The protective function of skin depends on successful completion of a tightly regulated multi-step differentiation program, during which the induction of markers for a specific stage in epidermal differentiation is coupled to repression of markers expressed at the preceding stage. We have explored the role of protein kinase C (PKC) in this process using an in vitro model system, in which cultures of primary mouse epidermal keratinocytes are induced to terminally differentiate by raising the Ca2+ concentration in the medium from 0.05 to 0.12 mM. At doses which activate PKC, 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleoyl-2-acetylglycerol block Ca(2+)-mediated induction of the spinous cell markers keratins K1 and K10 at both the protein and mRNA level. TPA and 1-oleoyl-2-acetylglycerol also rapidly repress K1 and K10 mRNA expression when added to differentiating keratinocyte cultures already expressing these markers. The inhibition of K1 mRNA expression by TPA is blocked in cells where PKC has been inactivated with bryostatin. TPA-mediated loss of K1 mRNA is also blocked in cells exposed to cycloheximide or actinomycin D implicating a PKC-induced protein factor in this process. The loss of K1 mRNA in TPA-treated cultures is the result of both a selective destabilization of K1 transcripts and a rapid inhibition of K1 gene transcription. In contrast to the dramatic repression of mRNAs typical for spinous cell differentiation, activation of PKC concurrently enhances expression of mRNAs and proteins for the granular cell markers loricrin and filaggrin. This response does not occur in cells pre-treated with bryostatin to inactivate PKC. Our results suggest that PKC is a fundamental regulator of the coordinate changes in keratinocyte gene expression that occur during the spinous to granular cell transition in epidermis.

Increased expression of cytokeratin and ferritin-H genes in tumorigenic clones of the SW 613-S human colon carcinoma cell line

Subclones of the SW 613-S human colon carcinoma cell line differ by their ability to induce tumors in nude mice and by their level of amplification of the c- myc gene. Clones with a high level of amplification are tumorigenic in nude mice whereas those with a low level are not. Genes overexpressed in the tumorigenic clones as compared to the nontumorigenic ones were searched by differential screening of a cDNA library. Two cDNA clones corresponding to cytokeratin K18 and ferritin-H chain were isolated. The steady state level of the corresponding mRNAs is higher in cells of all tumorigenic clones. The level of cytokeratin K8 mRNA, the specific partner of cytokeratin K18 in intermediate filaments of epithelial cells, is also elevated in these cells. For all three genes, this is mainly due to an increase in the transcription rate, as shown by a nuclear run-on assay. Immunoblotting experiments showed that cytokeratins K8, K18, and K19 are more abundant in cells of tumorigenic clones. The mRNA of the other subunit of apoferritin (ferritin-L chain) is expressed at the same level in both types of clones. The mRNAs of cytokeratins K18 and K8 and of ferritin-H chain are also overexpressed in cells of nontumorigenic clones which have acquired a tumorigenic phenotype after transfection of c- myc gene copies.

Induction of Calbindin-D 28K by 1,25-dihydroxyvitamin D 3 in cultured chicken intestinal cells

Intestinal cells from chicken embryos were grown in chemically defined, serum-free medium. The majority of cultured cells exhibits an epithelial-like morphology. As demonstrated by indirect immunofluorescence, the epithelial cells, and not the contaminating fibroblasts, express Calbindin-D 28K only after 1,25-dihydroxyvitamin D 3, the hormonally active form of vitamin D, is added to the culture medium. The highly sensitive reverse transcriptase-polymerase chain reaction shows that both Calbindin-D 28K mRNA and the corresponding primary unprocessed transcripts (pre-mRNA) are dramatically increased in cultured intestinal cells treated with 1,25-dihydroxyvitamin D 3, thus indicating that Calbindin-D 28K is induced by the increased rate of transcription of the corresponding gene.

Calbindin-D 28K-containing neurons in animal models of neurodegeneration: possible protection from excitotoxicity

Brain levels of the calcium binding protein Calbindin-D 28K (CaBP 28K) and CaBP 28K mRNA were measured for various animal models of neurodegenerative diseases (MPTP-treated C57BL/6J mice and Sprague-Dawley rats receiving striatal/intraperitoneal kainic acid or quinolinic acid into the nucleus basalis magnocellularis). Brain areas were tested (radioimmunoassay, Western blot, slot blot, and Northern blot) for levels of CaBP 28K and CaBP 28K mRNA. The various models did not exhibit any changes in protein or mRNA levels from the controls, suggesting that CaBP 28K-containing neurons were not lost after exposure to these neurotoxins. Immunocytochemical characterization of the substantia nigra of the MPTP-treated mice revealed that there was significant dopaminergic cell loss in this brain area after MPTP treatment. The majority of dopaminergic neurons that degenerated did not contain CaBP 28K. The small percentage of surviving neurons were CaBP 28K-positive. These results suggest that the presence of CaBP 28K may protect neurons from calcium-mediated neurotoxicity.

Mapping of the duplicated rabbit immunoglobulin kappa light chain locus.

The rabbit has two isotypic forms of the immunoglobulin kappa light chain, K1 and K2, which probably arose by duplication. In the normal rabbit, only traces of K2 light chains are produced. However, K2 levels are elevated in allotype-suppressed rabbits and in the Basilea strain which does not produce K1 because of a K1 mRNA splice site mutation. Previous cloning and sequencing showed that each isotype has its own set of J kappa genes but it was not known whether the two isotypes utilize shared or separate sets of V kappa genes. In addition, although genetic linkage of allotypes associated with the K1 and K2 genes has been demonstrated, physical linkage had not been previously demonstrated by overlapping cosmid or phage clones. We used pulsed field and transverse alternating field electrophoresis to obtain megabase maps and to estimate the size of the duplication of the rabbit kappa light chain locus. We found that the two C kappa genes are about 1 megabase apart. One explanation for the poor expression of K2, could be great physical distance from V kappa genes. However, we found that there are V kappa, J kappa and C kappa 2 genes within a approximately 105-kb fragment. Thus, physical distance of V kappa from C kappa 2 may not be the basis for poor K2 expression.

Detection of calbindin-D 28k mRNA in rat vestibular ganglion neurons by in situ hybridization

The cellular distribution of calbindin-D 28k mRNA in the rat vestibular ganglion was examined by in situ hybridization. Using a [ 35S]cDNA probe a neuronal subpopulation expressing calbindin-D 28k mRNA with a strong intensity has been identified. These findings confirm the presence of a subclass of calbindin-immunoreactive neurons in the rat vestibular ganglion.

Vitamin D-independent expression of chick brain calbindin-D 28K

A combination of calbindin-D 28K-specific cDNA probes and polyclonal antisera were used to investigate expression of the calbindin-D 28K in the vitamin D-deficient avian brain in vivo in response to pharmacological doses of the vitamin D 3 metabolite, 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3). Serum calcium levels were stimulated (2-fold) and intestinal calbindin-D 28K expression (between 10- and 30-fold) by 1,25(OH) 2D 3 (6.5 nmol/animal) after 12 h. In marked contrast, steady-state whole brain levels of calbindin-D 28K as judged by enzyme-linked immunoassay (ELISA) remained constant. Northern gel analysis revealed that three species of calbindin-D 28K mRNA (2.0, 2.6 and 3.1 kb) were present a priori in the vitamin D-deficient chick brain and that administration of pharmacological doses (6.5 nmol/animal) of 1.25(OH) 2D 3 failed to influence their relative abundance. Separate but parallel dot blot hybridization analyses also confirmed that brain calbindin-D 28K-mRNA levels were not influenced by 1,25(OH) 2D 3. These experiments demonstrate at the molecular level that, in contrast to the intestine, the gene encoding calbindin-D 28K in the brain is regulated by mechanism(s) or factors which are independent of vitamin D status.

Regulation of intestinal calbindin-D 28K gene expression: A solution hybridization study

A solution hybridization assay employing specific synthetic oligodeoxynucleotide probes was developed to study the regulation of intestinal calbindin-D 28K mRNA. The technique is rapid and quantitative and eliminates the need for sample transfer, blotting, autoradiography, and densitometry. Following validation of the assay, chick intestinal calbindin-D and calbindin-D mRNA levels were compared under conditions known to stimulate intestinal calcium (Ca) transport. Both the protein and its mRNA were undetectable in 25-day-old vitamin D-deficient chicks. Following acute administration of vitamin D 3, calbindin-D mRNA levels increased somewhat more rapidly than calbindin-D protein, but overall, the correlation was excellent. Chicks fed a nutritionally adequate diet for 14 days and then changed to a low Ca (0.1%) diet responded with increased calbindin-D and calbindin-D mRNA levels. Again the correlation was excellent over the ensuing 14-day experimental period. The combined effects of vitamin D repletion and dietary Ca status were also investigated with respect to calbindin D and its mRNA. Fourteen-day-old vitamin D-deficient chicks were changed to diets containing vitamin D and either adequate (1.2%) or low (0.3%) in Ca. The intestinal responses were measured at intervals up to 14 days. In the normal Ca situation, there were initial increases in both calbindin mRNA levels, which peaked at between 4 and 7 days, and calbindin protein levels, which peaked at 7 days. Both values subsequently declined during the remaining 7 days of the experimental period. In the low Ca situation, there were similar increases in calbindin mRNA and protein levels through 4 and 7 days respectively, but these levels remained high for the remainder of the 14-day experimental period. The present results demonstrate that intestinal tissue levels of calbindin D and its mRNA respond similarly to vitamin D repletion and dietary Ca restriction as well as the combination of these stimuli. There is no evidence to support significant post-transcriptional regulation of calbindin-D by Ca.

Calcium binding protein (calbindin-D 28k) gene expression in the developing and aging mouse cerebellum

Calbindin-D 28k (CaBP 28k) protein and gene expression were examined in the mouse cerebellum during development and aging utilizing slot and Northern blot hybridization analyses for mRNA levels, Western blot analysis and radioimmunoassay (RIA) for protein levels, and by in situ studies using immunocytochemistry and hybridization cytochemistry on prepared tissue sections. Samples were obtained and analyzed from C57BL/6J mice aged day of birth and postnatal weeks 1, 2, 4, 8, and 120. A specific cDNA and antibody for CaBP 28k were utilized in these studies. Analysis of mRNA levels showed a steady rise in CaBP 28k mRNA from birth to a peak at postnatal week (3.4-fold increase) and then a decline to steady-state levels at postnatal weeks 4 and 8 (47% reduction of peak level) followed by a reduction of CaBP 28k mRNA to birth levels at postnatal week 120. The specificity of the changes observed was tested by reprobing blots with β-actin cDNA. Analysis of CaBP 28k protein levels by both Western blot and RIA showed a similar pattern. In situ analysis of CaBP 28k mRNA levels, based on hybridization signal (silver grains per cell), demonstrated a rise in cellular CaBP 28K mRNA levels which peaked at postnatal week 2 (416.9 ± 52.1) and then declined to steady-state levels by postnatal weeks 4 and 8 (267.4 ± 35.8). Cellular CaBP 28k mRNA levels exhibited a dramatic reduction in the aged cerebellum (postnatal week 120; 78.3 ± 16.0). The levels of cellular CaBP 28k mRNA corresponded to the intensity of immunoreactive CaBP 28k localized by immunocytochemistry. The results are consistent with the hypothesis that CaBP 28k may play a critical role in Purkinje cell maturation and maintenance. Decreased CaBP 28k gene expression in the aged cerebellum is not simply a consequence of cell loss (remaining viable cells exhibit reduced CaBP 28k gene expression). Thus, CaBP 28k may have a neuroprotective and maintaining the concentration of CaBP 28k in Purkinje cells may be important for their survival.

Identification of Two Genes in the Unique Short Region of Pseudorabies Virus; Comparison with Herpes Simplex Virus and Varicella-zoster Virus

We have determined the nucleotide sequence of two genes in the unique short region of the genome of pseudorabies virus (PRV). Near the internal repeat, upstream of the gene encoding glycoprotein gX, we identified an open reading frame (ORF) encoding a protein of 390 amino acids. We designated this gene PK because the predicted protein contains most of the conserved motifs of a eukaryotic protein kinase. The protein shares amino acid homology with the protein kinases encoded by gene US3 of herpes simplex virus type 1 (HSV-1) and gene 66 of varicella-zoster virus. Near the terminal repeat, downstream of a gene encoding an 11K protein, we identified an ORF encoding a protein of 256 amino acids. We designated this gene 28K, the Mr of the predicted protein. Part of the amino acid sequence of 28K is homologous to the predicted US2 protein of HSV-1. Northern blot analysis revealed a 2.7 kb mRNA encoding the putative protein kinase and a 1.2 kb mRNA encoding the 28K protein in PRV-infected cells. The 5' ends of the mRNAs were mapped by primer extension. Two transcriptional start sites were identified for the PK mRNA: a minor start site immediately upstream of the ORF and a major start site (greater than 95% of the mRNA) within the ORF, 64 nucleotides upstream of an internal ATG codon. A single transcriptional start site was identified for the 28K mRNA immediately upstream of the ORF. Immunoblot analysis with anti-peptide sera revealed that, in cells infected with PRV, the PK gene was translated into two proteins with Mrs of 53K and 41K, and the 28K gene into a single protein with an Mr of 28K.

The expression of two kallikrein gene family members in the rat kidney

The mRNAs for two kallikrein gene family members expressed in the rat kidney have been characterized. One mRNA (PS) has previously been found in the pancreas and submaxillary gland and encodes true kallikrein. The second mRNA (Kl) encodes a novel kallikrein-like enzyme expressed in the kidney and submaxillary gland that retains many of the key amino acid residues for the characteristic enzymatic cleavage specificity of kallikrein. Two oligonucleotide hybridization probes specific for the K1 mRNA demonstrate that the K1 mRNA is expressed in the kidney and submaxillary gland, but in none of the other eight tissues known to express one or more members of the rat kallikrein gene family. The K1 mRNA is the dominant kallikrein-related mRNA of the kidney, expressed at roughly 10 times the level of the true kallikrein (PS) mRNA. In the submaxillary gland the K1 mRNA is expressed at roughly one-fourth the level of true kallikrein mRNA.

Tissue-specific regulation of the expression of rat kallikrein gene family members by thyroid hormone

We have altered the thyroid hormonal status of both male and female rats and examined the expression of six functional members of the rat kallikrein gene family (PS, S1, S2, S3, K1 and P1) in the submandibular gland (SMG), kidney, prostate, testis and anterior pituitary gland (AP) of these animals. On Northern-blot analysis with gene-specific oligonucleotide probes, the steady-state mRNA levels of S1, S2, S3, K1 and P1 were all dramatically altered in the SMG of male and female rats treated with propylthiouracil (PTU; 100 mg/litre of drinking water) or thyroxine (T4; 10 micrograms/100 mg body wt.) for 3 weeks. The SMG mRNA levels of these five genes were all lowered (30-90%) in hypothyroid (PTU-treated) male and female rats and elevated (1.4-4-fold, male; 1.5-11-fold, female) in the hyperthyroid (T4-treated) and PTU/T4-treated animals. In contrast, PS (true kallikrein) mRNA levels in the male or female SMG or kidney were essentially unchanged. K1 mRNA levels in the kidney were considerably less responsive to thyroid status than those in the SMG. Changes in S3 and P1 mRNA levels in the prostate were also variable, but essentially unaffected by these treatments. AP PS mRNA levels were also unaffected by changes in thyroid-hormonal status, as were levels of a novel P1-like mRNA in the testis. In summary, these studies demonstrate that the same kallikrein gene family member(s) may be differentially regulated by thyroid hormones in the rat SMG, kidney, prostate and pituitary, and thus further extend the concept of tissue-specific expression and hormonal regulation of the kallikrein gene family in the rat.

Differential regulation of keratin 8 and 18 messenger RNAs in differentiating F9 cells

F9 embryonal carcinoma cells (F9EC) can be induced to differentiate in vitro into epithelial cells expressing keratin 8 (K8) and keratin 18 (K18). cDNAs corresponding to K8 and K18 mRNAs were cloned and used to study the change in the abundance of these mRNAs during differentiation of F9 cells into parietal endoderm-like cells by treatment with retinoic acid (RA) or with RA and dibutyryl cAMP (Bt 2cAMP). Using an RNase protection assay, it was determined that K8 mRNA was induced slightly before K18 mRNA and that it accumulated to a greater extent than K18 mRNA. Furthermore, differentiation in presence of Bt 2cAMP plus RA resulted in an earlier induction of the two mRNAs and a higher level of expression of K8 mRNA. These results indicate that K8 and K18 mRNAs are regulated differently in F9 cells.

Differential expression of the epidermal K1 and K10 keratin genes during mouse embryo development

Induction of genes coding for the K1 and K10 keratins during mouse development was studied by measuring the accumulation of their respective mRNAs in day 10 to 17 embryos using an RNase protection assay. Although these two keratins are coexpressed in the suprabasal layers of the epidermis, it was found that while K1 mRNA was detectable as soon as day 10, K10 mRNA was not detectable before day 12. The expression of these genes at this stage of development was not expected since they are specifically associated with keratinization, a process that does not begin before day 17 of gestation. Histological examination of the epidermis of day 10 to 17 embryos suggests that both genes are induced in cells committed to epidermal differentiation, after stratification has started but before the onset of keratinization. It was also found that the two mRNAs increased in abundance steadily and significantly until day 16 and that, in spite of the expectation that filaments should contain equivalent amounts of each subunit, K1 mRNA remained more abundant than K10 mRNA at all times including in adult epidermis. These observations indicate that the two genes are regulated independently during development.

Increases in calbindin D 28K mRNA in the uterus of the domestic fowl induced by sexual maturity and shell formation

Uterine concentrations of calbindin D 28K mRNA were measured in immature pullets and laying hens by dot-blot hybridization using a [ 32P]cRNA probe prepared from the calbindin cDNA. In immature pullets, estrogen increased the calbindin mRNA level and the plasma concentration of 1,25-dihydroxyvitamin D 3 [1,25-(OH) 2D 3]. When testosterone was administered with estrogen there was a further increase in calbindin and its mRNA and an increase in the free 1,25-(OH) 2D 3 index calculated as the ratio of the molar concentrations of total 1,25-(OH) 2D 3 and vitamin D-binding protein (DBP). In laying hens the uterine concentration of calbindin mRNA was low 4 hr after ovulation, but increased most markedly 12 and 18 hr later, when shell calcification took place. Calbindin concentration remained unchanged during the different stages of egg formation but was much higher in laying hens than in pullets treated with sex steroids. Suppression of shell formation by premature expulsion of the egg decreased the concentrations of calbindin mRNA and uterine calbindin and the free 1,25-(OH) 2D 3 index in the plasma. A concomitant increase in calbindin and its mRNA was observed at resumption of shell formation in hens previously laying shell-less eggs. With-drawal of food for 44 hr decreased the uterine concentration of calbindin and its mRNA without a change in the free 1,25-(OH) 2D 3 index in the blood. It is concluded that the synthesis of uterine calbindin is stimulated primarily at sexual maturity and at calcification of the first shell by transcriptional processes. The daily increase in calbindin mRNA associated with shell formation and the absence of a concomitant change in calbindin concentration suggest that post-transcriptional processes exist and that stimuli other than the sex steroid or the 1,25-(OH) 2D 3 are involved in regulation of calbindin synthesis in the uterus.

Tissue-specific regulation of the concentration of Calbindin D 28K mRNA in the developing chicken

A BamHI-HindIII restriction fragment containing the 5′-terminal portion of the gene encoding chicken Calbindin D 28K was sequenced and used as a probe in Northern-blot hydridizations to RNA extracted from the brain and intestine of chickens at various stages of development. In both tissues Calbindin D 28K mRNA consists of a family of three species, which differ by size. In the intestine Calbindin D 28K mRNAs appear at hatching and reach a peak at day 7. In the brain the same RNA species are easily detected at least 7 days before hatching, show a moderate increase at hatching and remain essentially constant during the first 10 days of adult life. The concentration of Calbindin D 28K mRNAs in the intestine is strictly dependent on Vitamin D, while it is not in the cerebellum.

Cloning of the Human Keratin 18 Gene and Its Expression in Nonepithelial Mouse Cells

Human keratin 18 (K18) and the homologous mouse protein, Endo B, are intermediate filament subunits of the type I keratin class. Both are expressed in many simple epithelial cell types including trophoblasts, the first differentiated cell type to appear during mouse embryogenesis. The K18 gene was identified and cloned from among the 15 to 20 similar sequences identified within the human genome. The identity of the cloned gene was confirmed by comparing the sequence of the first two exons to the K18 cDNA sequence and transfecting the gene into various murine cell lines and verifying the encoded protein as K18 by immunoprecipitation and partial peptide mapping. The transfected K18 gene was expressed in mouse HR9 parietal endodermal cells and mouse fibroblasts even though the fibroblasts fail to express endogenous Endo B. S1 nuclease protection analysis indicated that mRNA synthesized from the transfected K18 gene is initiated at the same position as authentic K18 mRNA found in both BeWo trophoblastoma cells and HeLa cells. Pulse-chase experiments indicated that the human K18 protein is stable in murine parietal endodermal cells (HR9) which express EndoA, a complementary mouse type II keratin. Surprisingly, however, K18 was degraded when synthesized in cells which lack a type II keratin. This turnover of K18 may be an important mechanism by which epithelial cells maintain equal molar amounts of both type I and II keratins. In addition, the levels of the endogenous type I Endo B in parietal endodermal cells were compensatingly down regulated in the presence of the K18 protein, while the levels of the endogenous type II Endo A were not affected in any of the transfected cell lines.

Cloning of the human keratin 18 gene and its expression in nonepithelial mouse cells.

Human keratin 18 (K18) and the homologous mouse protein, Endo B, are intermediate filament subunits of the type I keratin class. Both are expressed in many simple epithelial cell types including trophoblasts, the first differentiated cell type to appear during mouse embryogenesis. The K18 gene was identified and cloned from among the 15 to 20 similar sequences identified within the human genome. The identity of the cloned gene was confirmed by comparing the sequence of the first two exons to the K18 cDNA sequence and transfecting the gene into various murine cell lines and verifying the encoded protein as K18 by immunoprecipitation and partial peptide mapping. The transfected K18 gene was expressed in mouse HR9 parietal endodermal cells and mouse fibroblasts even though the fibroblasts fail to express endogenous Endo B. S1 nuclease protection analysis indicated that mRNA synthesized from the transfected K18 gene is initiated at the same position as authentic K18 mRNA found in both BeWo trophoblastoma cells and HeLa cells. Pulse-chase experiments indicated that the human K18 protein is stable in murine parietal endodermal cells (HR9) which express EndoA, a complementary mouse type II keratin. Surprisingly, however, K18 was degraded when synthesized in cells which lack a type II keratin. This turnover of K18 may be an important mechanism by which epithelial cells maintain equal molar amounts of both type I and II keratins. In addition, the levels of the endogenous type I Endo B in parietal endodermal cells were compensatingly down regulated in the presence of the K18 protein, while the levels of the endogenous type II Endo A were not affected in any of the transfected cell lines.