Transcriptional Regulation of Jaw Osteoblasts: Development to Pathology.

Craniofacial and jaw bones have unique physiological specificities when compared to axial and appendicular bones. However, the molecular profile of the jaw osteoblast (OB) remains incomplete. The present study aimed to decipher the bone site-specific profiles of transcription factors (TFs) expressed in OBs in vivo. Using RNA sequencing analysis, we mapped the transcriptome of confirmed OBs from 2 different skeletal sites: mandible (Md) and tibia (Tb). The OB transcriptome contains 709 TF genes: 608 are similarly expressed in Md-OB and Tb-OB, referred to as "OB-core"; 54 TF genes are upregulated in Md-OB, referred to as "Md-set"; and 18 TF genes are upregulated in Tb-OB, referred to as "Tb-set." Notably, the expression of 29 additional TF genes depends on their RNA transcript variants. TF genes with no previously known role in OBs and bone were identified. Bioinformatics analysis combined with review of genetic disease databases and a comprehensive literature search showed a significant contribution of anatomical origin to the OB signatures. Md-set and Tb-set are enriched with site-specific TF genes associated with development and morphogenesis (neural crest vs. mesoderm), and this developmental imprint persists during growth and homeostasis. Jaw and tibia site-specific OB signatures are associated with craniofacial and appendicular skeletal disorders as well as neurocristopathies, dental disorders, and digit malformations. The present study demonstrates the feasibility of a new method to isolate pure OB populations and map their gene expression signature in the context of OB physiological environment, avoiding in vitro culture and its associated biases. Our results provide insights into the site-specific developmental pathways governing OBs and identify new major OB regulators of bone physiology. We also established the importance of the OB transcriptome as a prognostic tool for human rare bone diseases to explore the hidden pathophysiology of craniofacial malformations, among the most prevalent congenital defects in humans.

Dysregulated expression of the T cell cytokine Eta-1 in CD4-8- lymphocytes during the development of murine autoimmune disease.

The development of autoimmune disease in the MRL/MpJ-lpr inbred mouse strain depends upon the maturation of a subset of T lymphocytes that may cause sustained activation of immunological effector cells such as B cells and macrophages. We tested the hypothesis that abnormal effector cell activation reflects constitutive overexpression of a T cell cytokine. We found that a newly defined T cell cytokine, Eta-1, is expressed at very high levels in T cells from MRL/l mice but not normal mouse strains and in a CD4-8- 45R+ T cell clone. The Eta-1 gene encodes a secreted protein that binds specifically to macrophages, possibly via a cell adhesion receptor, resulting in alterations in the mobility and activation state of this cell type (Patarca, R., G. J. Freeman, R. P. Singh, et al. 1989. J. Exp. Med. 170:145; Singh, R. P., R. Patarca, J. Schwartz, P. Singh, and H. Cantor. 1990. J. Exp. Med. 171:1931). In addition, recent studies have indicated that Eta-1 can enhance secretion of IgM and IgG by mixtures of macrophages and B cells (Patarca, R., M. A. Lampe, M. V. Iregai, and H. Cantor, manuscript in preparation). Dysregulation of Eta-1 expression begins at the onset of autoimmune disease and continues throughout the course of this disorder. Maximal levels of Eta-1 expression and the development of severe autoimmune disease reflect the combined contribution of the lpr gene and MRL background genes.

Regulation of epidermal differentiation by a Distal-less homeodomain gene.

The Distal-less-related homeodomain gene Dlx3 is expressed in terminally differentiated murine epidermal cells. Ectopic expression of this gene in the basal cell layer of transgenic skin results in a severely abnormal epidermal phenotype and leads to perinatal lethality. The basal cells of affected mice ceased to proliferate, and expressed the profilaggrin and loricrin genes which are normally transcribed only in the latest stages of epidermal differentiation. All suprabasal cell types were diminished and the stratum corneum was reduced to a single layer. These data indicate that Dlx3 misexpression results in transformation of basal cells into more differentiated keratinocytes, suggesting that this homeoprotein is an important regulator of epidermal differentiation.

Coiled-coil trigger motifs in the 1B and 2B rod domain segments are required for the stability of keratin intermediate filaments.

Many alpha-helical proteins that form two-chain coiled coils possess a 13-residue trigger motif that seems to be required for the stability of the coiled coil. However, as currently defined, the motif is absent from intermediate filament (IF) protein chains, which nevertheless form segmented two-chain coiled coils. In the present work, we have searched for and identified two regions in IF chains that are essential for the stability necessary for the formation of coiled-coil molecules and thus may function as trigger motifs. We made a series of point substitutions with the keratin 5/keratin 14 IF system. Combinations of the wild-type and mutant chains were assembled in vitro and in vivo, and the stabilities of two-chain (one-molecule) and two-molecule assemblies were examined with use of a urea disassembly assay. Our new data document that there is a region located between residues 100 and 113 of the 2B rod domain segment that is absolutely required for molecular stability and IF assembly. This potential trigger motif differs slightly from the consensus in having an Asp residue at position 4 (instead of a Glu) and a Thr residue at position 9 (instead of a charged residue), but there is an absolute requirement for a Glu residue at position 6. Because these 13 residues are highly conserved, it seems possible that this motif functions in all IF chains. Likewise, by testing keratin IF with substitutions in both chains, we identified a second potential trigger motif between residues 79 and 91 of the 1B rod domain segment, which may also be conserved in all IF chains. However, we were unable to find a trigger motif in the 1A rod domain segment. In addition, many other point substitutions had little detectable effect on IF assembly, except for the conserved Lys-23 residue of the 2B rod domain segment. Cross-linking and modeling studies revealed that Lys-23 may lie very close to Glu-106 when two molecules are aligned in the A(22) mode. Thus, the Glu-106 residue may have a dual role in IF structure: it may participate in trigger formation to afford special stability to the two-chain coiled-coil molecule, and it may participate in stabilization of the two-molecule hierarchical stage of IF structure.

The homeoprotein DLX3 and tumor suppressor p53 co-regulate cell cycle progression and squamous tumor growth.

Epidermal homeostasis depends on the coordinated control of keratinocyte cell cycle. Differentiation and the alteration of this balance can result in neoplastic development. Here we report on a novel DLX3-dependent network that constrains epidermal hyperplasia and squamous tumorigenesis. By integrating genetic and transcriptomic approaches, we demonstrate that DLX3 operates through a p53-regulated network. DLX3 and p53 physically interact on the p21 promoter to enhance p21 expression. Elevating DLX3 in keratinocytes produces a G1-S blockade associated with p53 signature transcriptional profiles. In contrast, DLX3 loss promotes a mitogenic phenotype associated with constitutive activation of ERK. DLX3 expression is lost in human skin cancers and is extinguished during progression of experimentally induced mouse squamous cell carcinoma (SCC). Reinstatement of DLX3 function is sufficient to attenuate the migration of SCC cells, leading to decreased wound closure. Our data establish the DLX3-p53 interplay as a major regulatory axis in epidermal differentiation and suggest that DLX3 is a modulator of skin carcinogenesis.

The temporal and spatial expression of the novel Ca++-binding proteins, Scarf and Scarf2, during development and epidermal differentiation.

During the process of epidermal differentiation, intracellular and extracellular calcium (Ca++) concentrations induce an array of signaling pathways . Keratinocytes follow a complex Ca++-dependent program of differentiation moving from the basal proliferative layer, through the spinous and granular differentiated layers to ultimately culminate in the formation of the cornified layer of the epidermis. Members of the Ca++-binding proteins play a central role in the transduction of Ca++ signals. Utilizing a suppressive subtractive hybridization screen comparing basal and differentiated keratinocytes, we identified the novel Ca++-binding protein genes, Scarf (skin Calmodulin-related factor) and Scarf2, which have homology to calmodulin (CaM). In this study, we present a comprehensive analysis of the expression pattern for Scarf and Scarf2 transcripts and proteins in the developing mouse. To examine Scarf2 expression during embryogenesis, we performed in situ hybridization, and detected expression in the hair follicle, skin and nasal epithelium. These results showed substantial overlap with the previously reported Scarf gene expression [Hwang, M., Morasso, M.I., 2003. The novel murine Ca2+-binding protein, Scarf, is differentially expressed during epidermal differentiation. J. Biol. Chem. 278, 47827-47833]. Comparing the expression patterns of Scarf and Scarf2 proteins in neonatal and adult mouse skin with several structural epidermal proteins, i.e. keratin 14 (K14), keratin 1 (K1), loricrin (LOR) and filaggrin (FIL) showed that their expression overlaps K1, an early marker of keratinocyte differentiation. Interestingly, Scarf and Scarf2 were also detected in the tongue and oral epithelia, rib bone undergoing ossification and in the medullar region of thymus.

Developmental expression of the homeobox protein Distal-less 3 and its relationship to progesterone production in mouse placenta.

Distal-less 3 (Dlx3) is a homeobox factor that functions as a placental-specific transcriptional regulator. Dlx3 null mice (-/-) have compromised placental development and do not survive in utero past embryonic day (E) 9.5. The current studies were undertaken to examine the expression of Dlx3 in mouse placenta during gestation, and to determine whether Dlx3 was involved in placental progesterone production. Dlx3 was not detectable at E8.5 but was detected in E9.5 placenta with continuing but diminished expression through E15.5. Dlx3 immuno-localization was restricted to the labyrinth, was nuclear and was found in cytokeratin-positive cells. Previous studies in choriocarcinoma cell lines support the conclusion that Dlx3 is required for expression of 3'-hydroxysteroid dehydrogenase VI (3betaHSD VI), an obligate enzyme in the production of progesterone by trophoblast giant cells. In a rat trophoblast stem cell line (Rcho-1), Dlx3 expression was non-detectable in Rcho-1 cells induced to differ-entiate using mitogen withdrawal. In vitro progesterone production in placental cultures and 3betaHSD VI mRNA from Dlx3 (+/+), (+/-) and (-/-) mice were equivalent. In situ hybridization for 3betaHSD VI revealed mRNA expression restricted to trophoblast giants cells with no detectable expression in the labyrinth suggesting that Dlx3 and 3betaHSD VI were not colocalized within the placenta. These studies support the conclusion that Dlx3 protein expression is restricted to the labyrinth region of the murine placenta into late gestation and that Dlx3 does not appear to be expressed in trophoblast giant cells. Further, loss of Dlx3 was not correlated with synthesis of progesterone from E9.5 mouse placentas.

Differential expression of a Distal-less homeobox gene Xdll-2 in ectodermal cell lineages.

Neural induction in Xenopus requires the activation of new sets of genes that are necessary for cellular and regional specification of the neural tube. It has been reported earlier that members of the Distal-less homeobox gene family are specifically activated in distinct regions of the central nervous system (CNS) of Xenopus embryos (Dirksen et al., 1993; Papalopulu and Kintner, 1993). In this paper we describe in detail a Xenopus homeobox containing gene Xdll-2, which belongs to the Distal-less gene family. In contrast to other previously described Xenopus family members, Xdll-2 is expressed in the embryonic ectoderm and is specifically repressed in the CNS. This repression can be mimicked in isolated animal caps by treatment with activin. Expression of Xdll-2 persists in the epidermis and some neural crest cells. Because of its spatial and temporal expression pattern this gene is a good candidate to have a regulatory function in the initial formation of the epidermis. Its high level of expression in adult skin indicates that its function is continuously required in this tissue.

Phosphorylation of murine homeodomain protein Dlx3 by protein kinase C.

The Dlx3 homeodomain gene is expressed in terminally differentiated murine epidermal cells. As demonstrated for differentiation-specific granular markers, Dlx3 is activated in primary mouse keratinocytes cultured in vitro by increasing the level of the extracellular Ca(2+). This activation is mediated through a protein kinase C-dependent (PKC) pathway. In this study, we investigated whether PKC can modulate the activity of murine Dlx3 protein. Using in vitro kinase assays, we show that PKC enzymes phosphorylate the Dlx3 protein. Using keratinocyte nuclear extracts for the kinase reaction, we determined that Dlx3 protein is phosphorylated, and the phosphorylation is inhibited by the PKC-specific inhibitor GF109203X, suggesting that Dlx3 is phosphorylated by PKC in vivo. Of the PKC isoforms present in the epidermis, we tested alpha, delta, epsilon and zeta. Dlx3 is primarily phosphorylated by PKC alpha. By deletion and mutational analysis, we show that the serine residue S(138), located in the homeodomain of Dlx3 protein, was specifically phosphorylated by PKC. The phosphorylation of purified Dlx3 proteins by PKC partially inhibited formation of complexes between Dlx3 protein and DNA. These results suggest that Dlx3 protein can be directly phosphorylated by PKC and this affects the DNA binding activity of Dlx3.

Effect of mixing on the bioavailability of nitrofurantoin capsules.

The effect of blending time on the "in vivo" absorption of powder mixtures of similar composition, containing nitrofurantoin (NF), both in crystalline (10-50 u diameter) and macrocrystalline (70-80 u diameter) forms, was studied. The blending operations were performed using a Twin-Shell Blender (V-Type). The ingredients were blended long enough to obtain homogeneity, and this point was considered as "zero time". The mixing was continued and a intensifier bar was placed inside the blender. Samples were collected at 0; 30 and 45 minutes of blending and hard gelatin capsules were filled with these samples. A bioavailability study was performed in six healthy male volunteers. The experiments were conducted in a crossover fashion and each volunteer took one capsule of each time of blending of both microcrystalline and macrocrystalline nitrofurantoin. Urine samples were collected after the administration of the drug, and assayed by a spectro-fluorometric method. The total nitrofurantoin (NF) amount excreted in urine showed statistically significant differences at different times of blending only when capsules containing microcrystalline drug were administered.

DLX3 regulates bone mass by targeting genes supporting osteoblast differentiation and mineral homeostasis in vivo.

Human mutations and in vitro studies indicate that DLX3 has a crucial function in bone development, however, the in vivo role of DLX3 in endochondral ossification has not been established. Here, we identify DLX3 as a central attenuator of adult bone mass in the appendicular skeleton. Dynamic bone formation, histologic and micro-computed tomography analyses demonstrate that in vivo DLX3 conditional loss of function in mesenchymal cells (Prx1-Cre) and osteoblasts (OCN-Cre) results in increased bone mass accrual observed as early as 2 weeks that remains elevated throughout the lifespan owing to increased osteoblast activity and increased expression of bone matrix genes. Dlx3OCN-conditional knockout mice have more trabeculae that extend deeper in the medullary cavity and thicker cortical bone with an increased mineral apposition rate, decreased bone mineral density and increased cortical porosity. Trabecular TRAP staining and site-specific Q-PCR demonstrated that osteoclastic resorption remained normal on trabecular bone, whereas cortical bone exhibited altered osteoclast patterning on the periosteal surface associated with high Opg/Rankl ratios. Using RNA sequencing and chromatin immunoprecipitation-Seq analyses, we demonstrate that DLX3 regulates transcription factors crucial for bone formation such as Dlx5, Dlx6, Runx2 and Sp7 as well as genes important to mineral deposition (Ibsp, Enpp1, Mepe) and bone turnover (Opg). Furthermore, with the removal of DLX3, we observe increased occupancy of DLX5, as well as increased and earlier occupancy of RUNX2 on the bone-specific osteocalcin promoter. Together, these findings provide novel insight into mechanisms by which DLX3 attenuates bone mass accrual to support bone homeostasis by osteogenic gene pathway regulation.

The Dlx3 protein harbors basic residues required for nuclear localization, transcriptional activity and binding to Msx1.

The murine Dlx3 protein is a putative transcriptional activator that has been implicated during development and differentiation of epithelial tissue. Dlx3 contains a homeodomain and mutational analysis has revealed two regions, one N-terminal and one C-terminal to the homeodomain, that act as transcriptional activators in a yeast one-hybrid assay. In addition to transactivation, data are presented to demonstrate specific DNA binding and an association between Dlx3 and the Msx1 protein in vitro. Immunohistochemical analysis confirmed coexpression of Dlx3 and Msx1 proteins in the differentiated layers of murine epidermal tissues. Transcription factor function requires nuclear localization. In this study, the intracellular localization of the green fluorescent protein fused to Dlx3 was examined in keratinocytes induced to differentiate by calcium and is shown to localize to the nucleus. A bipartite nuclear localization signal (NLS) was identified by mutational analysis and shown to be sufficient for nuclear localization. This was demonstrated by insertion of the Dlx3 bipartite NLS sequence into a cytoplasmic fusion protein, GFP-keratin 14, which functionally redirected GFP-keratin 14 expression to the nucleus. Further analysis of Dlx3 NLS mutants revealed that the Dlx3 NLS sequences are required for specific DNA binding, transactivation potential and interactions with the Msx1 protein.

Regulation of the Dlx3 homeobox gene upon differentiation of mouse keratinocytes.

The Distal-less Dlx3 homeodomain gene is expressed in terminally differentiated murine epidermal cells, and there is evidence to support an essential role as a transcriptional regulator of the terminal differentiation process in these cells. In an attempt to determine the factors that induce Dlx3 gene expression, we have cloned the 1.2-kilobase pair proximal region of murine gene and analyzed its cis-regulatory elements and potential trans-acting factors. The proximal region of the Dlx3 gene has a canonical TATA box and CCAAT box, and the transcription start site was located 205 base pairs upstream from the initiation of translation site. Serial deletion analysis showed that the region between -84 and -34 confers the maximal promoter activity both in undifferentiated and differentiated primary mouse keratinocytes. Gel retardation assays and mutational analysis demonstrated that the transcriptional regulator NF-Y (also referred to as CBF) binds to a CCAAT box motif within this region and is responsible for the majority of the Dlx3 promoter activity. In addition, an Sp1-binding site was located immediately upstream of transcription start site that acts as a positive regulatory element of the Dlx3 promoter, independent of the CCAAT box motif. Importantly, elements residing between +30 to +60 of the Dlx3 gene are responsible for the Ca(2+)-dependent induction of Dlx3 during keratinocyte differentiation.

The homeodomain gene Xenopus Distal-less-like-2 (Xdll-2) is regulated by a conserved mechanism in amphibian and mammalian epidermis.

Xenopus Distal-less-like-2 (Xdll-2) is a gene encoding a homeodomain protein expressed predominantly in the epidermis of frog embryos. We report here that this epidermal expression is specified by a regulatory 5' flanking DNA region located within 933 bp of the start of transcription. This regulatory DNA also confers upon a globin reporter gene calcium-inducible expression in cultured murine keratinocytes and induction-dependent repression in frog ectodermal cells treated in vitro with activin A. These results reveal a new example of phylogenetically conserved, tissue-specific transcriptional regulation of a homeodomain gene.

A Xenopus distal-less gene in transgenic mice: conserved regulation in distal limb epidermis and other sites of epithelial-mesenchymal interaction.

In this paper, we show the conserved regulation of the homeodomain gene Distal-less-3 (Dlx-3) by analyzing the expression of a promoter from the Xenopus ortholog, Xdll-2, in transgenic mice. A 470-bp frog regulatory sequence confers appropriate expression on a lacZ reporter gene in the ectodermal component of structures derived from epithelial-mesenchymal interactions. Remarkably, this includes structures absent in Xenopus, such as the hair follicle and mammary gland, suggesting that conserved regulatory elements can be used to control the formation of structures peculiar to individual species. In addition, expression of Dlx-3 in developing limbs is highest at the most distal portion. This pattern is duplicated by the Xenopus promoter, indicating that this DNA may include sequences responsive to conserved proximodistal patterning signals in the vertebrate limb.

Cell adhesion regulates melanoma specific differentiation and interactions with the 3' region of the tyrosinase gene.

Decreased attachment to substratum has now been found to increase melanosome formation and cell-cell interaction in B16 melanoma. Since melanosome formation involves tyrosinase gene expression, we assayed for differential RNA expression by hybridization with probes from the distal ends of this gene, detecting unequal reactivity only with the 3' end probe. The same DNA showed binding of 2 nuclear proteins of 50 and 60 kd in unanchored cells, in contrast with a decreased binding of the 60 kd species, in nuclear extracts from attached cells. No comparable differences were detected with a gamma-actin DNA of identical length, suggesting that the changes observed are sequence-specific. Our studies suggest that the adhesion-mediated modulation of pigmentation in B16 melanoma correlates with differential macromolecular interactions with the 3' end of the tyrosinase gene.

A role for the homeobox protein Distal-less 3 in the activation of the glycoprotein hormone alpha subunit gene in choriocarcinoma cells.

Synthesis and secretion of chorionic gonadotropin in trophoblast cells of the placenta is required for establishment of early pregnancy in primates. Chorionic gonadotropin is a heterodimeric glycoprotein hormone consisting of alpha and beta subunits. Regulation of the alpha subunit gene within the placenta requires an array of cis elements within the 5'-flanking region of the promoter. Within this array of elements, the junctional regulatory element (JRE) putatively binds a placental-specific transcription factor. The aim of our studies was to determine the identity and role of the transcriptional regulator that binds to the JRE in choriocarcinoma cells (JEG3 cells). Mutations within the JRE resulted in reduction in basal expression of an alpha subunit reporter gene, suggesting that the JRE binding factor was necessary for full basal activity. Using electrophoretic mobility shift assays, we determined that the JRE was capable of serving as a homeobox factor-binding site. The homeobox factor, Distal-less 3 (Dlx 3) was found to be expressed in JEG3 cells and in the trophoblast layer of human chorionic villus but not in a gonadotrope cell line that also expresses the alpha subunit gene. Electrophoretic mobility shift assays revealed that recombinant Dlx 3 could bind specifically to the JRE and endogenous Dlx 3 was present in JRE/JEG3 nuclear protein complexes. Overexpression of Dlx 3 resulted in activation of an alpha subunit reporter gene. A JRE mutation resulted in attenuated activation of the alpha subunit reporter via an adjacent cis element, suggesting that JRE/Dlx 3 interactions may facilitate regulation of the alpha subunit gene at sites immediately upstream of the JRE. Our studies support the conclusion that Dlx 3 is a placental-specific transcriptional regulator that binds to the JRE and contributes to expression of the alpha subunit gene in cells of trophoblast origin.

Changes in cytoskeleton-associated molecules in cells with different degrees of proliferation and metastatic ability.

An antiserum prepared against the Triton-insoluble cytoskeleton of in vivo grown B16 melanoma tumor has been used to analyze the differential expression of cytoskeleton-associated molecules in cells with different degrees of proliferation and metastatic ability. This antiserum identified a major 97 kd molecule associated with the cytoskeletal fraction in B16 melanoma tumors, mouse embryo and in proliferating lymphocytes, with no reactivity with the 97 kd species in non proliferating lymphocytes. The antiserum revealed immune reactivity with a 180 kd Triton-insoluble species in normal adult mouse liver and kidney. A comparison of tumor cells with differing metastatic ability also showed a minor 180 kd component in poorly metastatic cells which appeared decreased and partly degraded in its more invasive counterpart. The differential recognition of a 97 kd species in resting and proliferating lymphocytes, as well as the different cleavage of a 180 kd species in tumor cells of differing metastatic ability, implies a role for these molecules in cell proliferation. The fact that these differences can be detected with an antiserum to tumor cell cytoskeleton suggests that this Triton-insoluble fraction may be a good source of molecules involved in growth control.

Influence of alcohol consumption on erythromycin ethylsuccinate kinetics.

The effect of ethyl alcohol ingestion on erythromycin kinetics was studied. Nine healthy volunteers, four males and five females, participated in the study. They received, in two separate occasions, 500 mg of erythromycin ethylsuccinate ester given with water or with an alcoholic beverage. The antibiotic was assayed in plasma, using a microbiological method. Absorption and disposition parameters were calculated according to classical pharmacokinetic techniques. A longer lag time and a decrease in AUC were observed when the antibiotic was given with alcohol. The differences were statistically significant. It is likely that the effect of alcohol on gastric emptying could be responsible for the delayed absorption of the antibiotic.

Amoxicillin kinetics and ethanol ingestion.

The effect of ethyl alcohol ingestion on amoxicillin kinetics was studied. Eight healthy volunteers participated in the study. They received, in three separate occasions, a 500-mg dose of the antibiotic given with water, hydroalcoholic solution or pisco-sour. Blood samples were drawn at appropriate intervals. Amoxicillin was assayed using a microbiological method. Absorption and disposition parameters were calculated by classical pharmacokinetic techniques. There were no statistical significant differences in the rate of elimination of the antibiotic in the three experimental conditions. Ethanol affected significantly the absorption rate constant, lag time and t peak. On the other hand, Cmax and AUC were not significantly modified. Thus, it can be concluded that ethyl alcohol has influence on the rate but not on the extent of absorption.