Novel Genetic Determinants of Dental Maturation in Children.

Dental occlusion requires harmonious development of teeth, jaws, and other elements of the craniofacial complex, which are regulated by environmental and genetic factors. We performed the first genome-wide association study (GWAS) on dental development (DD) using the Demirjian radiographic method. Radiographic assessments from participants of the Generation R Study (primary study population, N1 = 2,793; mean age of 9.8 y) were correlated with ~30 million genetic variants while adjusting for age, sex, and genomic principal components (proxy for population stratification). Variants associated with DD at genome-wide significant level (P < 5 × 10-8) mapped to 16q12.2 (IRX5) (lead variant rs3922616, B = 0.16; P = 2.2 × 10-8). We used Fisher's combined probability tests weighted by sample size to perform a meta-analysis (N = 14,805) combining radiographic DD at a mean age of 9.8 y from Generation R with data from a previous GWAS (N2 = 12,012) on number of teeth (NT) in infants used as proxy of DD at a mean age of 9.8 y (including the ALSPAC and NFBC1966). This GWAS meta-analysis revealed 3 novel loci mapping to 7p15.3 (IGF2BP3: P = 3.2 × 10-8), 14q13.3 (PAX9: P = 1.9 × 10-8), and 16q12.2 (IRX5: P = 1.2 × 10-9) and validated 8 previously reported NT loci. A polygenic allele score constructed from these 11 loci was associated with radiographic DD in an independent Generation R set of children (N = 703; B = 0.05, P = 0.004). Furthermore, profiling of the identified genes across an atlas of murine and human stem cells observed expression in the cells involved in the formation of bone and/or dental tissues (>0.3 frequency per kilobase of transcript per million mapped reads), likely reflecting functional specialization. Our findings provide biological insight into the polygenic architecture of the pediatric dental maturation process.

Improvement of Storage Medium for Cultured Human Retinal Pigment Epithelial Cells Using Factorial Design.

Storage of human retinal pigment epithelium (hRPE) can contribute to the advancement of cell-based RPE replacement therapies. The present study aimed to improve the quality of stored hRPE cultures by identifying storage medium additives that, alone or in combination, contribute to enhancing cell viability while preserving morphology and phenotype. hRPE cells were cultured in the presence of the silk protein sericin until pigmentation. Cells were then stored for 10 days in storage medium plus sericin and either one of 46 different additives. Individual effects of each additive on cell viability were assessed using epifluorescence microscopy. Factorial design identified promising additive combinations by extrapolating their individual effects. Supplementing the storage medium with sericin combined with adenosine, L-ascorbic acid and allopurinol resulted in the highest cell viability (98.6 ± 0.5%) after storage for three days, as measured by epifluorescence microscopy. Flow cytometry validated the findings. Proteomics identified 61 upregulated and 65 downregulated proteins in this storage group compared to the unstored control. Transmission electron microscopy demonstrated the presence of melanosomes after storage in the optimized medium. We conclude that the combination of adenosine, L-ascorbic acid, allopurinol and sericin in minimal essential medium preserves RPE pigmentation while maintaining cell viability during storage.

The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway.

Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin's potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated.

Sox-4 messenger RNA is expressed in the embryonic growth plate and regulated via the parathyroid hormone/parathyroid hormone-related protein receptor in osteoblast-like cells.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) exert potent and diverse effects in cells of the osteoblastic and chondrocytic lineages. However, downstream mediators of these effects are characterized inadequately. We identified a complementary DNA (cDNA) clone encoding the 5' end of the transcription factor Sox-4, using a subtracted cDNA library enriched in PTH-stimulated genes from the human osteoblast-like cell line OHS. The SOX-4 gene is a member of a gene family (SOX and SRY) comprising transcription factors that bind to DNA through their high mobility group (HMG)-type binding domain, and previous reports have implicated Sox proteins in various developmental processes. In situ hybridization of fetal and neonatal mouse hindlimbs showed that Sox-4 messenger RNA (mRNA) was expressed most intensely in the zone of mineralizing cartilage where chondrocytes undergo hypertrophy, and by embryonic day 17 (ED17), after the primary ossification center was formed, its expression was detected only in the region of hypertrophic chondrocytes. Sox-4 mRNA was detected in osteoblast-like cells of both human and rodent origin. In OHS cells, physiological concentrations (10(-10)-10(-9) M) of human PTH 1-84 [hPTH(1-84)] and hPT(1-34), but not hPTH(3-84), stimulated Sox-4 mRNA expression in a time-dependent manner, indicating involvement of the PTH/PTHrP receptor. Sox-4 transcripts also were detected in various nonosteoblastic human cell lines and tissues, in a pattern similar to that previously reported in mice. The presence of Sox-4 mRNA in hypertrophic chondrocytes within the mouse epiphyseal growth plate at sites that overlap or are adjacent to target cells for PTH and PTHrP, and its strong up-regulation via activated PTH/PTHrP receptors in OHS cells, makes it a promising candidate for mediating downstream effects of PTH and PTHrP in bone.

Difference in strength of autonomously replicating sequences among repeats in the rDNA region of Saccharomyces cerevisiae.

The rDNA region of Saccharomyces cerevisiae contains 100-200 tandemly repeated copies of a 9 kb unit, each with a potential replication origin. In the present studies of cloned fragments from the region involved in the regulation of replication of rDNA, we detected differences in autonomously replicating sequence (ARS) activity for clones from the same yeast strain. One clone, which showed very low ARS activity, carried a point mutation, a C instead of T, in position 9 of the essential 11 bp consensus ARS as compared to clones carrying the normal 10-of-11-bp match to the consensus. The mutation could be traced back to genomic rDNA where it represents about one-third of the rDNA units in that strain. Differences in ARS activity have implications for understanding the regulation of replication of rDNA, and the ratio of active to inactive ARS in the rDNA region may be important for potential generation of extrachromosomal copies.

Vitamin A-sensitive tissues in transgenic mice expressing high levels of human cellular retinol-binding protein type I are not altered phenotypically.

The suggested function of cellular retinol-binding protein type I [CRBP(I)] is to carry retinol to esterifying or oxidizing enzymes. The retinyl esters are used in storage or transport, whereas oxidized forms such as all-trans or 9-cis retinoic acid are metabolites used in the mechanism of action of vitamin A. Thus, high expression of human CRBP(I) [hCRBP(I)] in transgenic mice might be expected to increase the production of retinoic acid in tissues, thereby inducing a phenotype resembling vitamin A toxicity. Alternatively, a vitamin A-deficient phenotype could also be envisioned as a result of an increased accumulation of vitamin A in storage cells induced by a high hCRBP(I) level. Signs of vitamin A toxicity or deficiency were therefore examined in tissues from transgenic mice with ectopic expression of hCRBP(I). Testis and intestine, the tissues with the highest expression of the transgene, showed normal gross morphology. Similarly, no abnormalities were observed in other tissues known to be sensitive to vitamin A status such as cornea and retina, and the epithelia in the cervix, trachea and skin. Furthermore, hematologic variables known to be influenced by vitamin A status such as the hemoglobin concentration, hematocrits and the number of red blood cells were within normal ranges in the transgenic mice. In conclusion, these transgenic mice have normal function of vitamin A despite high expression of hCRBP(I) in several tissues.

Retinol-induced secretion of human retinol-binding protein in yeast.

Retinol-binding protein (RBP) functions as a transporter for retinol (vitamin A) in plasma in higher eukaryotes. We have successfully expressed human RBP in Saccharomyces cerevisiae, and its secretion was found to be induced by retinol also in this lower eukaryote. Reduced induction of secretion by retinol in a temperature-sensitive sec18-1 mutant that is blocked in secretion at the restricted temperature suggests that as in mammalian cells, RBP can be released from the endoplasmic reticulum upon addition of retinol. Thus, the molecular mechanism involved in retinol-dependent secretion of RBP appears to be conserved in yeast, and this points to yeast as a putative model system for studying retinol-regulated secretion of RBP. RBP purified from yeast was found to be indistinguishable from RBP purified from human plasma in several functional assays.

Binding and cyclic AMP stimulation by N-terminally deleted human PTHs (3-84 and 4-84) in a homologous ligand receptor system.

We have produced in yeast two human parathyroid hormone (hPTH) analogs with amino-terminal deletions, hPTH(3-84) and hPTH(4-84), employing the mating factor alpha (MF alpha) expression system. The authenticity of the polypeptides was demonstrated by amino-terminal analysis, amino acid composition, and molecular mass analysis. In cells (LLC-PK1) transfected with the human PTH/parathyroid hormone-related protein (PTHrP) receptor, using [125I-Tyr36]chickenPTHrP(1-36)NH2 as radioligand, binding studies revealed dissociation constants at equilibrium (Kd) for hPTH(3-84) and hPTH(4-84) of 4.7 and 8.0 nM, respectively, only slightly higher than natural recombinant hPTH(1-84) Kd = 2.3 nM). In comparison, [Nle8,18,Tyr34]bovinePTH(3-34)NH2 and [Tyr36]cPTHrP(1-36)NH2 showed equal Kd's of 1.9 nM. Neither of the N-terminally deleted hPTH analogs showed any detectable stimulation of cAMP production in the cells at concentrations below 20 nM. At supersaturated concentrations (500 nM) with receptor occupancy of more than 95% these hPTH analogs revealed about 15% rest agonism compared with that of hPTH(1-84). hPTH(1-84) and [Tyr36]cPTHrP(1-36)NH2 showed an equal half maximal cyclic adenosine monophosphate (cAMP) stimulation of about 0.8 and 0.7 nM, respectively. The hPTH analogs did not show any ability to antagonize cellular cAMP production induced by either hPTH or [Tyr36]cPTHrP(1-36)NH2. [Nle8,18,Tyr34]bPTH(3-34)NH2 did also not antagonize cAMP stimulation by hPTH, but inhibited [Tyr36]cPTHrP(1-36)NH2-induced cAMP production by 40% when present at a 1000 M excess. These distinct results related to PTH and PTHrP from different species are important to consider in experiments evaluating potential hPTH or PTHrP antagonism, and employment of a hPTH/PTHrP receptor model is a requirement.

Retinyl ester storage is altered in liver stellate cells and in HL60 cells transfected with cellular retinol-binding protein type I.

It is suggested that cellular retinol-binding proteins are important for intracellular metabolism of retinol. Retinol bound to cellular retinol-binding proteins may be esterified with long chain fatty acids by the enzyme lecithin: retinol acyltransferase or may be oxidized to retinoic acid metabolites used in the mechanism of action of vitamin A. The aim of this present report was to determine whether altered levels of cellular retinol-binding protein type I influenced retinol storage and activation. Two different cell types have been examined after transfection with vectors producing sense or antisense mRNA for cellular retinol-binding protein type I. When HL60 cells were transfected with the expression vector for sense cellular retinol-binding protein type I high amounts of cellular retinol-binding protein type I mRNA and protein were produced. We observed that HL60 cells esterified less retinol than control cells without cellular retinol-binding protein type I. Cellular retinol-binding protein type I had, however, no effects on the proliferation or differentiation of HL60 cells by retinoids. Liver stellate cells transfected with the vector for sense cellular retinol-binding protein type I esterified more retinol than cells transfected with the expression vector for antisense cellular retinol-binding protein type I, while retinol esterification in control cells was intermediate. In conclusion, our data show that cellular retinol-binding protein type I influences retinol esterification both in liver stellate cells and in HL60 cells.

Retinyl ester storage is normal in transgenic mice with enhanced expression of cellular retinol-binding protein type I.

This report describes the production and characterization of transgenic mice with high expression of human cellular retinol-binding protein type I [hCRBP(I)]. In initial experiments, overexpression of hCRBP(I) was driven by the strong promoter SR(alpha), but no transgenic offspring were produced. When we used the less efficient mouse metallothionein I promoter fused to the hCRBP(I) cDNA for microinjection, we obtained 12% transgenic offspring. Two of these transgenic mice (409/1 and 401/2) expressed mRNA and immunoreactive hCRBP(I) in several organs. Both lines had relatively high contents of hCRBP(I) in intestine, testis and epididymis. On the other hand, only 401/2 transgenic mice had high contents of hCRBP(I) in kidney. Effects on storage of vitamin A were studied by measuring the concentration of retinyl esters in different organs. The concentrations of retinyl esters in liver, lung and kidney did not significantly differ between transgenic and control mice, and the concentration of total retinol in plasma was within the normal range in transgenic mice. Furthermore, feeding mice a diet with high or low concentrations of vitamin A for 2 wks resulted in no marked differences in the concentrations of retinyl esters in liver, kidney, lung, intestine and testis in transgenic mice compared with control mice. Therefore, in spite of high expression of hCRBP(I) in several organs, the transgenic mice had normal storage of retinyl esters in all organs studied. The present in vivo study indicates that the CRBP(I) content alone does not control retinyl ester storage.

The C-terminal RNLL sequence of the plasma retinol-binding protein is not responsible for its intracellular retention.

An in vitro model system using COS cells that transiently express human plasma retinol binding protein has been set up in which we are able to mimic the retinol dependent secretion of this protein observed in hepatocytes. In the absence of its ligand, plasma retinol binding protein is retained in the endoplasmic reticulum. It contains a C-terminal sequence, RNLL, that could function as a cryptic KDEL motif and thus be responsible for its retention in the endoplasmic reticulum. The model system has been used to test a mutant lacking these four last amino acids for retention and retinol induced secretion. The results obtained show that although plasma retinol binding protein is retained in the endoplasmic reticulum, the RNLL sequence does not seem to be responsible for its retention.

Isolation and characterization of two biologically active O-glycosylated forms of human parathyroid hormone produced in Saccharomyces cerevisiae. Identification of a new motif for O-glycosylation.

Expression and secretion of human parathyroid hormone in Saccharomyces cerevisiae were achieved by fusing a cDNA encoding the mature human parathyroid hormone (hPTH) to the preproregion of the yeast mating factor alpha. Purified hPTH from yeast-culture medium was found to contain, in addition to the native unglycosylated form, two mannosylated variants with different molecular masses. The three hPTH forms were processed identically, resulting in the same 84 amino acid polypeptides with amino acid sequences identical to the native hormone. In both the O-glycosylated forms that were separated by isocratic reverse-phase HPLC, two mannose-linked residues were localized to Thr79. In addition, the most glycosylated form showed a heterogeneous modification of three, four or five mannosyl residues linked at Ser66. Lysine is N-terminally located to Ser66 and probably stimulates this glycosylation, which introduces a possible new motif for O-glycosylation in yeast. The two glycosylated forms of hPTH had similar biological activity which was identical to the native form of hPTH in a hormone-sensitive adenylate cyclase assay in bone sarcoma cells. Thus, a C-terminal O-glycosylation of hPTH with up to seven mannosyl residues/molecule did not affect the biological activity of the hormone, making possible production of hPTH with potential different pharmacokinetic properties.

Characterization of a K26Q site-directed mutant of human parathyroid hormone expressed in yeast.

Human parathyroid hormone (hPTH) is susceptible to proteolytical cleavage both in humans and when expressed as a secretory product in Escherichia coli (Høgseth, A., Blingsmo, O. R., Saether, O., Gautvik, V. T., Holmgren, E., Hartmanis, M., Josephson, S., Gabrielsen, O. S., Gordeladze, J. O., Alestrøm, P., and Gautvik, K. M. (1990) J. Biol. Chem. 265, 7338-7344) and Saccharomyces cerevisiae (Gabrielsen, O. S., Reppe, S., Saether, O., Blingsmo, O. R., Sletten, K., Gordeladze, J. O., Høgset, A., Gautvik, V. T., Alestrøm, P., Oyen, T. B., and Gautvik, K. M. (1990) Gene (Amst.) 90, 255-262). In the latter system, one major site of cleavage was identified (Arg25-Lys26 decreased Lys27). To produce hPTH resistant to this proteolytic processing, a point mutation changing Lys26 to Gln was introduced, and the modified gene expressed in S. cerevisiae as a fusion protein with the alpha-factor leader sequence. The resulting major form of hPTH secreted to the growth medium was of full length showing that the mutation had eliminated internal processing. Consequently, the yield of the mutant hormone was significantly higher than obtained with the natural peptide. Using improved purification procedures, a significantly higher purity was also obtained. The secreted mutant hPTH-(1-84,Q26) had the correct size, full immunological reactivity with two different hPTH antisera, correct amino acid composition and N-terminal sequence, and correct mass as determined by mass spectrometry. Furthermore, the introduced mutation did not reduce the biological activity of the hormone as judged from its action in three biological assay systems: 1) a hormone-sensitive osteoblast adenylate cyclase assay; 2) an in vivo calcium mobilizing assay in rats; and 3) an in vitro bone resorption assay.

Efficient secretion of human parathyroid hormone by Saccharomyces cerevisiae.

A cDNA encoding mature human parathyroid hormone (hPTH) was expressed in Saccharomyces cerevisiae, after fusion to the prepro region of yeast mating factor alpha (MF alpha). Radioimmunoassay showed high levels of hPTH immunoreactive material in the growth medium (up to 10 micrograms/ml). More than 95% of the immunoreactive material was found extracellularly as multiple forms of hormone peptides. Three internal cleavage sites were identified in the hPTH molecule. The major cleavage site, after a pair of basic amino acids (aa) (Arg25Lys26 decreases Lys27), resembles that recognized by the KEX2 gene product on which the MF alpha expression-secretion system depends. The use of a protease-deficient yeast strain and the addition of high concentrations of aa to the growth medium, however, not only changed the peptide pattern, but also resulted in a significant increase in the yield of intact hPTH (1-84) (more than 20% of the total amount of immunoreactive material). The secreted hPTH (1-84) migrates like a hPTH standard in two different gel-electrophoretic systems, co-elutes with standard hPTH on reverse-phase high-performance liquid chromatography, reacts with two hPTH antibodies raised against different parts of the peptide, has a correct N-terminal aa sequence, and has full biological activity in a hormone-sensitive osteoblast adenylate cyclase assay.