Tissue-specific expression is conferred by a sequence from the 5'' end of the rat albumin gene.

We have constructed a transient expression vector containing 400 bp of rat albumin gene immediate 5'-flanking sequences inserted 5' to the bacterial enzyme chloramphenicol acetyl transferase (CAT). We have transfected various clones of rat hepatoma cells representing different states of expression of the liver phenotype with this vector (pALB-cat) and also with two control vectors containing viral promoters (pSVE-cat and pRSV-cat), and measured activity of the bacterial enzyme CAT in cellular extracts 48 h later. The albumin flanking sequences are able to direct highly efficient CAT expression, compared with the control vectors, only in cells which express their own albumin gene: the albumin-negative hepatoma cells are at least 100 times less efficient in expressing CAT after transfection with the pALB-cat plasmid than are the albumin-positive ones. An unexpected result of our study is the total inability of the rat albumin flanking sequences to direct expression in albumin-producing mouse hepatoma cells.     

Induction of specific human growth hormone binding sites in rat liver by human growth hormone.

125I-Labeled hGH was bound to liver plasma membranes which were obtained from female rats. The binding was displaced by hGH, hPRL, bPRL, rPRL and bGH but not by rGH. This result indicated that hGH was bound to lactogenic binding sites in rat livers. After hypophysectomy, the binding was markedly decreased. Treatment of hypophysectomized rats with hGH (80 micrograms/day) for 10 days increased the binding sites for hGH. These binding sites were different from those found in normal female rat livers because of their high affinity and specificity for hGH. These results indicate that hGH induces specific binding sites for hGH in rat livers.     

Tissue-specific and expression of porcine growth hormone gene in BAC transgenic mice

One of the primary goals of traditional livestock breeding is to improve growth rate and optimise body size. Growth rate can be significantly increased by integrating a growth hormone (GH) transgene under the control of a ubiquitous promoter, but while such animals do demonstrate increased growth there are also serious deleterious side-effects to the animals health. Here we report the generation and initial characterization of transgenic mice that carried a porcine BAC encoding the porcine GH gene. We show that GH expression is restricted specifically to the pituitary, is associated with elevated IGF-1 levels, and results in growth enhancement. No negative effects to the health of the transgenic animals were detected. This initial characterisation supports the use of BAC pGH transgene in livestock studies.     

Tissue-specific expression of the human alpha 1-antitrypsin gene is controlled by multiple cis-regulatory elements.

Human alpha 1-antitrypsin (AAT) is expressed in the liver, and a 318 bp fragment immediately flanking the CAP site of the gene was found to be sufficient to drive the expression of a reporter gene (CAT) specifically in hepatoma cells. The enhancing activity however, was orientation-dependent. The DNA fragment was separated into a distal region and a proximal region. A "core enhancer" sequence GTGGTTTC is present within the distal region and is capable of activity enhancement in both orientations when complemented by the proximal region in the sense orientation. The results strongly suggest that there are multiple cis-acting elements in the human AAT gene that confer cell specificity for its expression. Nuclear proteins prepared from the hepatoma cells bound specifically to the proximal region in a band-shifting assay that was resistant to competition by the globin promoter DNA. Foot-printing analysis showed a protected domain within the proximal region that contains a nearly perfect palindromic sequence TGGTTAATATTCACCA, which may be important in the regulation of AAT expression in the liver.     

Tissue-specific determinants of human atrial natriuretic factor gene expression in cardiac tissue

Elements controlling tissue-specific expression of the human atrial natriuretic factor gene have been examined in primary cultures of neonatal rat cardiocytes. When a 68-base pair fragment from human atrial natriuretic factor (hANF) 5'-flanking sequence (positions -400 to -333) was placed upstream from the herpes simplex thymidine kinase promoter linked to a bacterial reporter gene (chloramphenicol acetyltransferase), a tissue-specific positive regulatory effect was observed in atrial as well as ventricular cardiocytes but not in nonmyocardial cells. The cis-acting element in this fragment was orientation- and position-dependent. Examination of nuclear protein extracts for the presence of factors capable of interacting with the 5'-flanking sequence of the hANF gene revealed a cardiocyte-specific factor which bound to the 68-base pair fragment. This association was both tissue- and sequence-specific. These findings indicate that a cis-acting element present in the proximal 5'-flanking sequence confers tissue-specific expression upon the hANF gene, possibly through association with a cardiac-specific nuclear protein.     

Heparin binding epidermal growth factor-like growth factor is a transforming growth factor beta-regulated gene in intestinal epithelial cells.

Heparin binding epidermal growth factor-like growth factor (HB-EGF) is an EGF-related peptide with prominent effects on cell growth and migration. We explored potentially unique characteristics of HB-EGF in the intestinal epithelial cell line RIE-1. HB-EGF stimulated [(3)H]thymidine incorporation to a level equivalent to transforming growth factor alpha (TGFalpha). HB-EGF also rapidly activated MAPK and induced cyclin D1 in mid-G1 with kinetics similar to TGFalpha. Unlike TGFalpha, HB-EGF mRNA was induced within 1 h by a variety of stimuli, including TGFbeta1. Maximal induction by TGFbeta (7-fold) occurred within 2 h of treatment. Actinomycin D decay curves showed that TGFbeta1 had no effect on HB-EGF mRNA half-life (T(1/2) 20 min). Induction of HB-EGF by TGFbeta1 was not affected by pretreatment with the MEK inhibitor PD-98059 while inhibition of protein kinase C either partially (calphostin C) or completely (staurosporin) blocked induction. Our results suggest that major differences exist in the regulation of the closely related EGF family members TGFalpha and HB-EGF. TGFbeta and HB-EGF, structurally unrelated peptides with potent effects on wound healing, may function coordinately to mediate responses to wounding or cell injury in the intestinal epithelium.     

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.     

Shear-induced platelet-derived growth factor gene expression in human endothelial cells is mediated by protein kinase C.

Our previous studies have shown that steady shear stress causes a transient increase of platelet-derived growth factor (PDGF) A and B chain mRNA levels in human umbilical vein endothelial cells (HUVEC). In the present study, we elucidated the signaling pathway of shear stress in HUVEC by examining the roles of protein kineses, intracellular calcium, cyclooxygenase, and guanine nucleotide-binding proteins (G proteins) in the PDGF gene induction by shear. The protein kinase C inhibitors, H7 and staurosporine, strongly inhibited the shear-induced PDGF gene expression in HUVEC. In contrast, HA1004, a cAMP- and cGMP-dependent protein kinases inhibitor, was only slightly inhibitory. BAPTA/AM, an intracellular calcium chelator, partially (50%) inhibited the shear-induced PDGF gene expression. The cyclooxygenase inhibitors, ibuprofen and indomethacin, were slightly inhibitory. A 35-50% inhibition of shear-induced PDGF gene expression was found with GDP-beta-S, an inhibitor of G proteins. These results suggest that shear-induced PDGF gene expression in HUVEC is mainly mediated by protein kinase C activation and requires intracellular calcium. Furthermore, G proteins seem to be involved in this process, whereas prostaglandin synthesis via cyclooxygenase pathway is not. We propose a mechanism of shear-induced PDGF gene expression in HUVEC: Shear stress, either directly or indirectly (G protein-mediated), enhances the membrane phosphoinositide turnover via phospholipase C, producing diacylglycerol, an activator of protein kinase C. The activated protein kinase C then triggers the subsequent PDGF gene expression.     

Pleiotrophin gene expression is highly restricted and is regulated by platelet-derived growth factor.

Pleiotrophin (PTN) is a growth and neurite extension promoting polypeptide which is highly expressed in brain and in tissues derived from mesenchyme. The PTN gene is developmentally regulated and is closely related to the MK and RI-HB genes, both of which are developmentally regulated and induced by retinoic acid. We now have screened 17 cell lines and report that expression of the PTN gene in these cells is restricted to embryo fibroblasts and intestinal smooth muscle cells. However, NIH 3T3 cells stimulated by the platelet-derived growth factor (PDGF) express a marked increase in levels of PTN mRNA whereas retinoic acid failed to increase levels of PTN mRNA in NIH 3T3 cells or in F9 embryonal carcinoma cells within 72 hours of exposure. The results suggest that expression of the PTN gene is highly restricted and that the PTN gene is a new member of the PDGF-induced cytokine family.