In long bones, morphogenesis is followed by an extended period of growth. Longitudinal growth occurs at the growth plate by endochondral ossification, a process in which cartilage is formed and then remodeled into bone (1, 2). The multistep process of chondrogenesis involves: 1) the aggregation of mesenchymal cells into prechondrogenic condensations, 2) differentiation of the condensed prechondrogenic mesenchyme into small immature chondrocytes, and 3) chondrocyte maturation followed by ossification, starting in the center of the element.Bone morphogenetic proteins (BMPs) 1 play an important role in skeletal development (3-5). At the early stage of bone development, BMP-2 and BMP-4 are expressed in specific areas of the limb bud 3 and misexpression of noggin, a glycoprotein that blocks actions of BMP-2 and BMP-4, prior to the onset of chondrogenesis leads to the total absence of skeletal elements (6), suggesting that BMP signaling is absolutely required for prechondrogenic condensations. At the late stage, BMP-2, expressed in the growth plate of long bone, accelerates longitudinal bone growth by stimulating growth plate chondrocyte proliferation and hypertrophy (7,8).To understand the regulatory mechanism of BMP-2 gene, we have previously characterized the BMP-2 promoter and identified control elements in osteoblasts and chondrocytes in BMP-2 gene (9). BMP-2 autoregulates its own expression through its proximal promoter (10) in a similar fashion that the decapentaplegic (dpp) gene is regulated in drosophila. dpp is a drosophila homologue of BMP-2. In drosophila, expression of dpp is regulated by dorsal, a homologue of mammalian NF-B (11, 12). In chick embryo, overexpression of a mutated form of I-B␣ protein, which blocks NF-B activation, leads to abnormality in limb bud development, and changes in downstream signals include BMP-4 (13). These findings suggest that NF-B may participate in the regulatory process of the BMP-2 gene.NF-B is a group of transcription factors including five members, p65, c-Rel, RelB, p50, and p52. They form homodimers or heterodimers to regulate expression of a variety of genes and are involved in many biological functions (14). It has been demonstrated previously that NF-B family members p50 and p52 are absolutely required for osteoclast development and p50/p52 double knock-out (dKO) mice failed to form osteoclasts and developed severe osteopetrosis (15).The role of NF-B in BMP-2 gene expression and functions of NF-B in other bone cells, such as chondrocytes, have not been reported. In the present studies, we have examined the regulatory mechanism of BMP-2 by NF-B in chondrocytes in vitro and in vivo. We searched the Ϫ2712/ϩ165 region of BMP-2 gene and found two putative NF-B response elements. Using different experimental approaches, we demonstrate that these NF-B response elements are functional in chondrocytes and NF-B positively regulates BMP-2 gene transcription through these response elements. We also show that in growth plate chondrocytes of 2-week-old NF-B p50/p52 dKO mice, BMP-...
A large percentage of human hepatomas produce a-fetoprotein (AFP), but the levels of AFP expression vary greatly among hepatomas. To understand the molecular basis for this variation, we analyzed tanscriptional regulatory activities associated with the 5'-flanking region of the AFP gene in two human hepatoma cell lines, HuH-7 and huH-1/cl-2, which produce a high and a low level of AFP, respectively. We found that the low level of AFP production in huH-1/cl-2 is due to the action of at least two silencer regions located between the enhancer and the promoter of the AFP gene. In contrast, no silencer activity is expressed in HuH-7. We identified 5'-CTTCATAACTAATACTT-3' to be a core sequence responsible for the negative regulatory activity. This sequence is repeated four times in a strong, distal silencer region, Sd, whereas one copy is present in a weak, proximal silencer region, Sp. The silencer reduces transcriptional initiation by blocking enhancer activation of the AFP promoter in a position-dependent manner. The silencer functions in the presence of positive transcription factors and may play a key role in developmental repression as well as variable expression of the AFP gene in hepatomas.The a-fetoprotein (AFP) gene is a developmentally regulated gene whose activity decreases rapidly after birth and becomes hardly detectable in adult life. However, the AFP gene is often reactivated to a high level in hepatocellular carcinoma (HCC) and teratocarcinomas (1, 4, 36). In contrast, the expression of the albumin gene, which is evolutionarily related to the AFP gene, steadily increases during normal development and stays high throughout adult life.Elevated levels of AFP have been observed in more than 70% of HCC patients, making AFP as a reliable diagnostic and prognostic marker of HCC. The AFP levels among AFP-positive patients, however, vary widely. Analysis of mechanisms underlying the variation of AFP production in HCC is of importance from clinical as well as basic biological standpoints.HuH-7 and huH-1/cl-2 are two human hepatoma cell lines that exhibit various liver-specific functions (23-25). HuH-7 produces a large amount of AFP and a moderate level of albumin. huH-1/cl-2, on the other hand, produces only a detectable amount of AFP but a high level of albumin. These cell lines provide useful model systems for the study of the molecular basis for different levels of expression of the AFP and albumin genes in hepatoma.We have shown previously that in HuH-7 cells, AFP gene expression is regulated positively by the enhancer, promoter, and glucocorticoid-responsive element (27,32,39). We have not so far detected DNA elements that suppress AFP gene transcription in this cell line. In this study, we analyzed mechanisms responsible for a low level of AFP gene expression in huH-1/cl-2. Two possibilities were considered: (i) the reduced activity of positive regulatory elements and (ii) the activation of negative control elements. We present evidence that the latter mechanism is largely * Corresponding author. t Presen...
Apolipoprotein Al (Apo Al) is the major protein component of high density lipoprotein (HDL) particles.HDL particles mediate the removal of cholesterol from extra-hepatic tissues via a process known as reverse cholesterol transport. Augmented production of Apo Al will likely be beneficial to those who suffer from the consequences of hypercholesterolemia. One approach to increase expression of the protein is to identify nuclear factor(s) that enhance Apo Al promoter activity. Therefore, we have used transient transfection to study a limited portion (-474 to -7) of the gene and showed that a cis-regulatory element, site C had a permissive effect on the ability of an adjacent site B to increase promoter activity by 30-fold. The importance of element C prompted us to Identify the factor(s) that interact with this site. Results showed that HNF-4, a new member of the thyroid/steroid hormone receptor superfamily interacts with site C to enhance activity of the promoter. Based on this observation and that of the known inhibitory effects of ARP-1 on site C, we postulate a model which may account for the tissue-specific expression of the rat Apo Al gene.
The induction of rat hepatic mRNA S11 by L-T3 (T3) is a useful model for studying the mechanisms of thyroid hormone action. Although numerous reports have examined the response of mRNA S11 to various physiological and hormonal manipulations, the role of S11 protein in cellular metabolism remains unknown. In this study we show that mRNA S11 is abundantly expressed and regulated by T3 only in liver and small intestine. High levels of the mRNA are present at birth, but drop sharply between 30-60 days of age. These and other features of the S11 gene product were similar to those of rat apolipoprotein-A1 (Apo-A1). The sequence of S11 cDNA was identical to a portion of the Apo-A1 mRNA, thus confirming identity of the S11 mRNA. To examine whether DNA sequences immediately adjacent to the transcription start site mediate the effects of thyroid hormone, we measured the activity of an Apo-A1 gene fragment, U-1 (-474 to -7) using a transient transfection assay. The activity of the full-length U-1 DNA in HuH-7 hepatoma cells was 2- to 2.5-fold higher in the presence of thyroid hormone. This finding closely matched previous results using the in vitro nuclear run-on assay. Internal deletion of a motif that resembles a thyroid hormone response element from U-1 DNA not only abolished the induction by T3, but suppressed promoter activity by 3- to 4-fold in response to the hormone.(ABSTRACT TRUNCATED AT 250 WORDS)
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