Expression of human cathepsin L or human cathepsin V in mouse thymus mediates positive selection of T helper cells in cathepsin L knock-out mice

A genetic deficiency of the cysteine protease cathepsin L (Ctsl) in mice results in impaired positive selection of conventional CD4+ T helper cells as a result of an incomplete processing of the MHC class II associated invariant chain or incomplete proteolytic generation of positively selecting peptide ligands. The human genome encodes, in contrast to the mouse genome, for two cathepsin L proteases, namely cathepsin L (CTSL) and cathepsin V (CTSV; alternatively cathepsin L2). In the human thymic cortex, CTSV is the predominately expressed protease as compared to CTSL or other cysteine cathepsins. In order to analyze the functions of CTSL and CTSV in the positive selection of CD4+ T cells we employed Ctsl knock-out mice crossed either with transgenic mice expressing CTSL under the control of its genuine human promoter or with transgenic mice expressing CTSV under the control of the keratin 14 (K14) promoter, which drives expression to the cortical epithelium. Both human proteases are expressed in the thymus of the transgenic mice, and independent expression of both CTSL and CTSV rescues the reduced frequency of CD4+ T cells in Ctsl-deficient mice. Moreover, the expression of the human cathepsins does not change the number of CD4+CD25+Foxp3+ regulatory T cells, but the normalization of the frequency of conventional CD4+ T cell in the transgenic mice results in a rebalancing of conventional T cells and regulatory T cells. We conclude that the functional differences of CTSL and CTSV in vivo are not mainly determined by their inherent biochemical properties, but rather by their tissue specific expression pattern.

Tissue-specific induction of EROD activity and CYP1A protein in Sparus aurata exposed to B(a)P and TCDD

The purpose of this study was to compare xenobiotic CYP1A induction in liver, gills, and excretory kidney of gilthead seabream, Sparus aurata. Fishes were exposed via water for 20 days to different concentrations of benzo(a)pyrene (B(a)P) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). CYP1A was measured at the enzyme activity level as 7-ethoxyresorufin-O-deethylase (EROD) activity, and at the protein level by means of ELISA. The liver displayed the highest absolute levels of EROD activity, both under non-exposed and exposed conditions. Organ- or inducer-related differences in the time course of CYP1A induction were moderate; however, the magnitude of the induction response varied between the organs and between B(a)P and TCDD. In the case of TCDD, liver, and kidney yielded a comparable induction response, whereas in the case of B(a)P, the kidney showed a substantially higher maximum induction factor than the liver. In the gills, the two xenobiotics resulted in similar maximum induction factors. In B(a)P-exposed seabream, EROD activities and CYP1A protein levels showed a good correlation in all three organs, whereas with TCDD as inducer the correlation was poor, what was mainly due to a decrease of EROD activities at the higher concentrations of TCDD, while CYP1A protein levels showed no concomitant decline. Overall, the study revealed both similarities and differences in the time-, concentration-, and inducer-dependent CYP1A responses of the three target organs, liver, kidney, and gills. Although, the findings of this study principally confirm the notion of the liver as the major metabolic organ in fish, they also provide evidence for substantial metabolic potential in gills and particularly in the kidney.

Plasma S-nitrosothiol status in neonatal calves: ontogenetic associations with tissue-specific S-nitrosylation and nitric oxide synthase

Neonatal cattle and in part neonates of other species have manyfold higher plasma concentrations of nitrite plus nitrate than mature cows and subjects of other species, suggesting an enhanced and needed activation of the nitric oxide (NO) axis at birth. While the biological half-life of NO is short (<1 sec), its functionality can be prolonged, and in many regards more discretely modulated, when it reacts with low-molecular-weight and protein-bound thiols to form S-nitrosothiols (RSNO), from which NO subsequently can be rereleased. We used the calf as a model to test the hypothesis that plasma concentrations of RSNO are elevated at birth in mammals, correlate with ascorbate and urate levels, are selectively generated in critical tissue beds, and are generated in a manner temporally coincident with changes in tissue levels of active NO synthases (NOS). Plasma concentrations of RSNO, ascorbate, and urate were highest immediately after birth (Day 0), dropped >50% on Day 1, and gradually decreased over time, reaching a nadir in mature cattle. Albumin and immunoglobulin G were identified as major plasma RSNO. The presence of S-nitrosocysteine (SNC, a validated marker for S-nitrosylated proteins), inducible NOS (iNOS), and activated endothelial NOS (eNOS phosphorylated at Ser1177) in different tissues was analyzed by immunohistochemistry in another group of similar-aged calves. SNC, iNOS, and phosphorylated eNOS were detected in liver and ileum at the earliest timepoint of sampling (4 hrs after birth), increased between 4 and 24 hrs, and then declined to near-nondetectable levels by 2 weeks of life. Our data show that the neonatal period in the bovine species is characterized by highly elevated and coordinated NO-generating and nitrosylation events, with the ontogenetic changes occurring in iNOS and eNOS contents in key tissues as well as RSNO products and associated antioxidant markers.

Novel functional profiling approach combining reverse phase protein microarrays and human 3-D ex vivo tissue cultures: expression of apoptosis-related proteins in human colon cancer

Cancer is caused by a complex pattern of molecular perturbations. To understand the biology of cancer, it is thus important to look at the activation state of key proteins and signaling networks. The limited amount of available sample material from patients and the complexity of protein expression patterns make the use of traditional protein analysis methods particularly difficult. In addition, the only approach that is currently available for performing functional studies is the use of serial biopsies, which is limited by ethical constraints and patient acceptance. The goal of this work was to establish a 3-D ex vivo culture technique in combination with reverse-phase protein microarrays (RPPM) as a novel experimental tool for use in cancer research. The RPPM platform allows the parallel profiling of large numbers of protein analytes to determine their relative abundance and activation level. Cancer tissue and the respective corresponding normal tissue controls from patients with colorectal cancer were cultured ex vivo. At various time points, the cultured samples were processed into lysates and analyzed on RPPM to assess the expression of carcinoembryonic antigen (CEA) and 24 proteins involved in the regulation of apoptosis. The methodology displayed good robustness and low system noise. As a proof of concept, CEA expression was significantly higher in tumor compared with normal tissue (p<0.0001). The caspase 9 expression signal was lower in tumor tissue than in normal tissue (p<0.001). Cleaved Caspase 8 (p=0.014), Bad (p=0.007), Bim (p=0.007), p73 (p=0.005), PARP (p<0.001), and cleaved PARP (p=0.007) were differentially expressed in normal liver and normal colon tissue. We demonstrate here the feasibility of using RPPM technology with 3-D ex vivo cultured samples. This approach is useful for investigating complex patterns of protein expression and modification over time. It should allow functional proteomics in patient samples with various applications such as pharmacodynamic analyses in drug development.

A specific expression profile of heat-shock proteins and glucose-regulated proteins is associated with response to neoadjuvant chemotherapy in oesophageal adenocarcinomas

BACKGROUND Oesophageal adenocarcinomas often show resistances to chemotherapy (CTX), therefore, it would be of high interest to better understand the mechanisms of resistance. We examined the expression of heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) in pretherapeutic biopsies of oesophageal adenocarcinomas to assess their potential role in CTX response. METHODS Ninety biopsies of locally advanced adenocarcinomas before platin/5-fluorouracil (FU)-based CTX were investigated by reverse phase protein arrays (RPPAs), immunohistochemistry (IHC) and quantitative RT-PCR. RESULTS CTX response strongly correlated with survival (P=0.001). Two groups of tumours with specific protein expression patterns were identified by RPPA: Group A was characterised by low expression of HSP90, HSP27 and p-HSP27((Ser15, Ser78, Ser82)) and high expression of GRP78, GRP94, HSP70 and HSP60; Group B exhibited the inverse pattern. Tumours of Group A were more likely to respond to CTX, resulting in histopathological tumour regression (P=0.041) and post-therapeutic down-categorisation from cT3 to ypT0-T2 (P=0.040). High HSP60 protein (IHC) and mRNA expression were also associated with tumour down-categorisation (P=0.016 and P=0.004). CONCLUSION Our findings may enhance the understanding of CTX response mechanisms, might be helpful to predict CTX response and might have translational relevance as they highlight the role of potentially targetable cellular stress proteins in the context of CTX response.

Aboral ectoderm-specific expression and regulation of the LpS1 genes of {\it Lytechinus pictus\/}

The Spec genes serve as molecular markers for examining the ontogeny of the aboral ectoderm lineage of sea urchin embryos. These genes are activated at late-cleavage stage only in cells contributing to the aboral ectoderm of Strongylocentrotus purpuratus and encode 14,000-17,000 Da calcium-binding proteins. A comparative analysis was undertaken to better understand the mechanisms underlying the activation and function of the Spec genes by investigating Spec homologues from Lytechinus pictus, a distantly related sea urchin. Spec antibodies cross-reacted with 34,000 Da proteins in L. pictus embryos that displayed a similar ontogenetic pattern to that of Spec proteins. One cDNA clone, LpS1, was isolated by hybridization to a synthetic oligonucleotide corresponding to a calcium-binding domain or EF-hand. The LpS1 mRNA has developmental properties similar to those of the Spec mRNAs. LpS1 encodes a 34,000 Da protein containing eight EF-hand domains, which share structural homology with the Spec EF-hands; however, little else in the protein sequence is conserved, implying that calcium-binding is important for Spec protein function. Genomic DNA blot analysis showed two LpS1 genes, LpS1$\alpha$ and LpS1$\beta$, in L. pictus. Partial gene structures for both LpS1$\alpha$ and $\beta$ were constructed based on genomic clones isolated from an L. pictus genomic library. These revealed internal duplications of the LpS1 genes that accounted for the eight EF-hand domains in the LpS1 proteins. Sequencing analysis showed there was little in common among the 5$\sp\prime$-flanking regions of the LpS1 and Spec genes except for the presence of a binding site for the transcription factor USF.^ A sea urchin gene-transfer expression system showed that 762 base pairs (bp) of 5$\sp\prime$-flanking DNA from the LpS1$\beta$ gene were sufficient for correct temporal and spatial expression of reporter genes in sea urchin embryos. Deletions at the 5$\sp\prime$ end to 511, 368, or 108bp resulted in a 3-4 fold decrease in chloramphenicol acetyltransferase (CAT) activity and disrupted the restricted activation of the lac Z gene in aboral ectoderm cells.^ A full-length Spec1 protein and a truncated LpS1 protein were induced and partially purified from an in vitro expression system. (Abstract shortened with permission of author.) ^

Molecular mechanisms of chondrocyte-specific expression on the mouse pro$\alpha$1(II) collagen gene

Type II collagen is a major chondrocyte-specific component of the cartilage extracellular matrix and it represents a typical differentiation marker of mature chondrocytes. In order to delineate cis-acting elements of the mouse pro$\alpha1$(II) collagen gene that control chondrocyte-specific expression in intact mouse embryos, we generated transgenic mice harboring chimeric constructions in which varying lengths of the promoter and intron 1 sequences were linked to a $\beta$-galactosidase reporter gene. A construction containing a 3000-bp promoter and a 3020-bp intron 1 fragment directed high levels of $\beta$-galactosidase expression specifically to chondrocytes. Successive deletions of intron 1 delineated a 48-bp fragment which targeted $\beta$-galactosidase expression to chondrocytes with the same specificity as the larger intron 1 fragment. When the Col2a1 promoter was replaced with a minimal $\beta$-globin promoter, the 48-bp intron 1 sequence was still able to target expression of the transgene to chondrocytes, specifically. Therefore a 48-bp intron 1 DNA segment of the mouse Col2a1 gene contains the necessary information to confer high-level, temporally correct, chondrocyte expression to a reporter gene in intact mouse embryos and that Col2a1 promoter sequences are dispensable for chondrocyte expression. Nuclear proteins present selectively in mouse primary chondrocytes and rat chondrosarcoma cells bind to the three putative HMG (High-Mobility-Group) domain protein binding sites in this 48-bp sequence and the chondrocyte-specific proteins likely bind the DNA through minor groove. Together, my results indicate that a 48-bp sequence in Col2a1 intron 1 controls chondrocyte-specific expression in vivo and suggest that chondrocytes contain specific nuclear proteins involved in enhancer activity. ^

Regulatory sequences of the porcine THBD gene facilitate endothelial-specific expression of bioactive human thrombomodulin in single- and multitransgenic pigs.

BACKGROUND Among other mismatches between human and pig, incompatibilities in the blood coagulation systems hamper the xenotransplantation of vascularized organs. The provision of the porcine endothelium with human thrombomodulin (hTM) is hypothesized to overcome the impaired activation of protein C by a heterodimer consisting of human thrombin and porcine TM. METHODS We evaluated regulatory regions of the THBD gene, optimized vectors for transgene expression, and generated hTM expressing pigs by somatic cell nuclear transfer. Genetically modified pigs were characterized at the molecular, cellular, histological, and physiological levels. RESULTS A 7.6-kb fragment containing the entire upstream region of the porcine THBD gene was found to drive a high expression in a porcine endothelial cell line and was therefore used to control hTM expression in transgenic pigs. The abundance of hTM was restricted to the endothelium, according to the predicted pattern, and the transgene expression of hTM was stably inherited to the offspring. When endothelial cells from pigs carrying the hTM transgene--either alone or in combination with an aGalTKO and a transgene encoding the human CD46-were tested in a coagulation assay with human whole blood, the clotting time was increased three- to four-fold (P<0.001) compared to wild-type and aGalTKO/CD46 transgenic endothelial cells. This, for the first time, demonstrated the anticoagulant properties of hTM on porcine endothelial cells in a human whole blood assay. CONCLUSIONS The biological efficacy of hTM suggests that the (multi-)transgenic donor pigs described here have the potential to overcome coagulation incompatibilities in pig-to-primate xenotransplantation.

Tissue-specific metabolism of benzo[a]pyrene in rainbow trout (Oncorhynchus mykiss): a comparison between the liver and immune organs.

Polycyclic aromatic hydrocarbons (PAHs) are immunotoxicants in fish. In mammals, phase I metabolites are believed to be critically involved in the immunotoxicity of PAHs. This mechanism has been suggested for fish as well. The present study investigates the capacity of immune organs (head kidney, spleen) of rainbow trout, Oncorhynchus mykiss, to metabolize the prototypic PAH, benzo[a]pyrene (BaP). To this end, we analyzed 1) the induction of enzymatic capacity measured as 7-ethoxyresorufin-O-deethylase (EROD) activity in immune organs compared with liver, 2) the organ profiles of BaP metabolites generated in vivo, and 3) rates of microsomal BaP metabolite production in vitro. All measurements were done for control fish and for fish treated with an intraperitoneal injection of 15 mg BaP/kg body weight. In exposed trout, the liver, head kidney, and spleen contained similar levels of BaP, whereas EROD induction differed significantly between the organs, with liver showing the highest induction factor (132.8×), followed by head kidney (38.4×) and spleen (1.4×). Likewise, rates of microsomal metabolite formation experienced the highest induction in the liver of BaP-exposed trout, followed by the head kidney and spleen. Microsomes from control fish displayed tissue-specific differences in metabolite production. In contrast, in BaP-exposed trout, microsomes of all organs produced the potentially immunotoxic BaP-7,8-dihydrodiol as the main metabolite. The findings from this study show that PAHs, like BaP, are distributed into immune organs of fish and provide the first evidence that immune organs possess inducible PAH metabolism leading to in situ production of potentially immunotoxic PAH metabolites.

siRNA mediated knockdown of tissue factor expression in pigs for xenotransplantation.

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi-transgenic pigs for xenotransplantation.

Molecular regulatory mechanisms and embryonic tissue specification in sea urchin development

To understand how a eukaryote achieves differential transcription of genes in precise spatial patterns, the molecular details of tissue specific expression of the Strongylocentrotus purpuratus Spec2a gene were investigated by functional studies of the cis-regulatory components in the upstream enhancer. Regional activation of Spec2a in the aboral ectoderm is conferred by a combination of activators and repressors. The positive regulators include previously identified SpOtx and a trans-regulatory factor binding at the CCAAT site in the Spec2a enhancer. The nuclear protein binding to the CCAAT box was determined to be the heterotrimeric CCAAT binding factor (SpCBF). SpCBF also mediates general activation in the ectoderm. The negative regulators consist of an oral ectoderm repressor (OER), an endoderm repressor (ENR), and an S. Purpuratus goosecoid homologue (SpGsc). OER functions to prevent expression in the oral ectoderm, while ENR is required to repress endoderm expression. SpGsc antagonizes the SpOtx function by competing for binding at SpOtx target genes in oral ectoderm, where it functions as an active repressor. Thus, SpOtx and SpGsc perform collectively to establish and maintain the oral-aboral axis. Finally, purification of ENR and OER proteins from sea urchin blastula stage nuclear extracts was performed using site-specific DNA-affmity chromatography. ^

Studies of the expression of DNA repair related genes

This dissertation examines the biological functions and the regulation of expression of DNA ligase I by studying its expression under different conditions.^ The gene expression of DNA ligase I was induced two- to four-fold in S-phase lymphoblastoid cells but was decreased to 15% of control after administration of a DNA damaging agent, 4-nitroquinoline-1-oxide. When cells were induced into differentiation, the expression level of DNA ligase I was decreased to less than 15% of that of the control cells. When the gene of DNA ligase I was examined for tissue specific expression in adult rats, high levels of DNA ligase I mRNA were observed in testis (8-fold), intermediate levels in ovary and brain (4-fold), and low levels were found in intestine, spleen, and liver (1- to 2-fold).^ In confluent cells of normal skin fibroblasts, UV irradiation induced the gene expression of DNA ligase I at 24 and 48 h. The induction of DNA ligase I gene expression requires active p53 protein. Introducing a vector containing the wild type p53 protein in the cells caused an induction of the DNA ligase I protein 24 h after the treatment.^ Our results indicate that, in addition to the regulation by phosphorylation/dephosphorylation, cellular DNA ligase I activity can be regulated at the gene transcription level, and the p53 tumor suppresser is one of the transcription factors for the DNA ligase I gene. Also, our results suggest that DNA ligase I is involved in DNA repair as well as in DNA replication.^ Also, as an early attempt to clone the human homolog of the yeast CDC9 gene which has been shown to be involved in DNA replication, DNA repair, and DNA recombination, we have identified a human gene with mRNA of 1.7 kb. This dissertation studies the gene regulation and the possible biological functions of this new human gene by examining its expression at different stages of the cell cycle, during cell differentiation, and in cellular response to DNA damage.^ The new gene that we recently identified from human cells is highly expressed in brain and reproductive organs (BRE). This BRE gene encodes an mRNA of 1.7-1.9 kb, with an open reading frame of 1,149 bp, and gives rise to a deduced polypeptide of 383 amino acid residues. No extensive homology was found between BRE and sequences from the EMBL-Gene Banks. BRE showed tissue-specific expression in adult rats. The steady state mRNA levels were high in testis (5-6 fold), ovary and brain (3-4 fold) compared to the spleen level, but low in intestine and liver (1-2 fold). The expression of this gene is responsive to DNA damage and/or retinoic acid (RA) treatment. Treatment of fibroblast cells with UV irradiation and 4-nitroquinoline-1-oxide caused more than 90% and 50% decreases in BRE mRNA, respectively. Similar decreases in BRE expression were observed after treatment of the brain glioma cell line U-251 and the promyelocytic cell line HL-60 with retinoic acid. (Abstract shortened by UMI). ^

The transcriptional regulation of the {\it neu\/} gene: Examples of activation, repression, and cell-specific expression

The neu oncogene encodes a growth factor receptor-like protein, p185, with an intrinsic tyrosine kinase activity. A single point mutation, an A to T transversion resulting in an amino acid substitution from valine to glutamic acid, in the transmembrane domain of the rat neu gene was found to be responsible for the transforming and tumorigenic phenotype of the cells that carry it. In contrast, the human proto-neu oncogene is frequently amplified in tumors and cell lines derived from tumors and the human neu gene overexpression/amplification in breast and ovarian cancers is known to correlate with poor patient prognosis. Examples of the human neu gene overexpression in the absence of gene amplification have been observed, which may suggest the significant role of the transcriptional and/or post-transcriptional control of the neu gene in the oncogenic process. However, little is known about the transcriptional mechanisms which regulate the neu gene expression. In this study, three examples are presented to demonstrate the positive and negative control of the neu gene expression.^ First, by using band shift assays and methylation interference analyses, I have identified a specific protein-binding sequence, AAGATAAAACC ($-$466 to $-$456), that binds a specific trans-acting factor termed RVF (for EcoRV factor on the neu promoter). The RVF-binding site is required for maximum transcriptional activity of the rat neu promoter. This same sequence is also found in the corresponding regions of both human and mouse neu promoters. Furthermore, this sequence can enhance the CAT activity driven by a minimum promoter of the thymidine kinase gene in an orientation-independent manner, and thus it behaves as an enhancer. In addition, Southwestern (DNA-protein) blot analysis using the RVF-binding site as a probe points to a 60-kDa polypeptide as a potential candidate for RVF.^ Second, it has been reported that the E3 region of adenovirus 5 induces down-regulation of epidermal growth factor (EGF) receptor through endocytosis. I found that the human neu gene product, p185, (an EGF receptor-related protein) is also down-regulated by adenovirus 5, but via a different mechanism. I demonstrate that the adenovirus E1a gene is responsible for the repression of the human neu gene at the transcriptional level.^ Third, a differential expression of the neu gene has been found in two cell model systems: between the mouse fibroblast Swiss-Webster 3T3 (SW3T3) and its variant NR-6 cells; and between the mouse liver tumor cell line, Hep1-a, and the mouse pancreas tumor cell line, 266-6. Both NR-6 and 266-6 cell lines are not able to express the neu gene product, p185. I demonstrate that, in both cases, the transcriptional repression of the neu gene may account for the lack of the p185 expression in these two cell lines. ^

Transcriptional regulation of cell lineage-specific expression of the murine type I collagen genes and of osteoblast differentiation

The aim of my project is to examine the mechanisms of cell lineage-specific transcriptional regulation of the two type I collagen genes by characterizing critical cis-acting elements and trans-acting factors. I hypothesize that the transcription factors that are involved in the cell lineage-specific expression of these genes may have a larger essential role in cell lineage commitment and differentiation. I first examined the proximal promoters of the proα1(I) and the proα2(I) collagen genes for cell type-specific DNA-protein interactions, using in vitro DNaseI and in vivo DMS footprinting. These experiments demonstrated that the cis-acting elements in these promoters are accessible to ubiquitous DNA-binding proteins in fibroblasts that express these genes, but not in other cells that do not express these genes. I speculate that in type I collagen-expressing cells, cell type-specific enhancer elements facilitate binding of ubiquitous proteins to the proximal promoters of these genes. Subsequently, examination of the upstream promoter of the proα(I) collagen gene by transgenic mice experiments delineated a 117 bp sequence (-1656 to -1540 bp) as the minimum element required for osteoblast-specific expression. This 117 bp element contained two segments that appeared to have different functions: (1) the A-segment, which was necessary to obtain osteoblast-specific expression and (2) the C-segment, which was dispensable for osteoblast-specific expression, but was necessary to obtain high-level expression. In experiments to identify trans-acting factors that bind to the 117 bp element, I have demonstrated that the cell lineage-restricted homeodomain proteins, Dlx2, Dlx5 and mHOX, bound to the A-segment and that the ubiquitous transcription factor, Sp1, bound to the C-segment of this element. These results suggested a model where the binding of cell lineage-restricted proteins to the A-segment and of ubiquitous proteins to the C-segment of the 117 bp element of the proα1 (I) collagen gene activated this gene in osteoblasts. These results, combined with additional evidence that Dlx2, Dlx5 and mHOX are probably involved in osteoblast differentiation, support my hypothesis that the transcription factors involved in osteoblast-specific expression of type I collagen genes may have essential role in osteoblast lineage commitment and differentiation. ^