Downstream Targets of Homeobox Gene HLX Show Altered Expression in Human Idiopathic Fetal Growth Restriction

Homeobox gene transcription factors play critical roles in normal placental development and are expressed in specialised trophoblast cells. Abnormal trophoblast cell function is associated with pregnancy disorders including fetal growth restriction. Our studies show homeobox gene HLX expression in trophoblast cells (1) and that HLX is significantly decreased in fetal growth restriction (2). HLX gene inactivation in cultured trophoblast cells shows that HLX is a regulator of cytokine-dependent trophoblast proliferation (3). Hepatocyte growth factor (HGF) activates trophoblast cell migration in a paracrine fashion and its receptor, c-met, is expressed in trophoblast cells. This study investigates the regulation of HGF/c-met mediated trophoblast migration by HLX, in two human trophoblast cell lines SGHPL-4 and HTR-8/SVNeo. HGF stimulation significantly increased HLX mRNA expression (e.g. 43.2 ± 2.5, HGF v. 18.4 ± 1.7 control, densitometric units, P < 0.001, n = 3). siRNA-mediated inactivation of HLX resulted in significantly decreased trophoblast migration (e.g. 32 ± 4, siRNA v. 127 ± 12 control, migrated cells, P < 0.05, n = 4). When HLX was inactivated in the presence of HGF stimulation, migration remained significantly decreased (e.g. 112 ± 15, siRNA + HGF v. 368 ± 44 HGF, migrated cells, P < 0.05, n = 4). In order to determine if HGF is acting via the c-met receptor, the Met tyrosine kinase inhibitor, SU11274, was employed to inhibit c-met activity. c-met inhibition resulted in significantly reduced HLX mRNA expression (e.g. 2.1 ± 0.32, SU11274 v. 12.3 ± 1.4 control, densitometric units, P < 0.05, n = 3). HLX expression remained significantly reduced with HGF stimulation and SU11274 mediated c-met inhibition (e.g. 8.02 ± 1.3, SU11274 v. 38.3 ± 5.4 HGF, densitometric units, P < 0.05, n = 3). This is the first study to show that homeobox gene HLX is a downstream effector gene of HGF, that HLX regulates trophoblast migration and that HGF, via its receptor c-met, acts through HLX to control cell migration. (1) Rajaraman G, Murthi P, Quinn L, Brennecke SP, Kalionis B. Homeodomain protein HLX is expressed primarily in cytotrophoblast cell types in the early human placenta. (2008) Reproduction, Fertility a (2) Murthi P, Doherty V, Said J, Donath S, Brennecke SP, Kalionis B. Homeobox gene HLX1 expression is decreased in idiopathic human fetal growth restriction. (2006) Am J Pathol. 2006 Feb;168(2):511–8. (3) Rajaraman G, Murthi P, Leo B, Brennecke SP, Kalionis B. Homeobox gene HLX1 is a regulator of colony stimulating factor-1 dependent cell proliferation. (2007) Placenta Volume 28, Issue 10, October

Download Full-text

205. Homeobox gene TGIF is increased in human idiopathic fetal growth restriction

Reproduction Fertility and Development ◽

10.1071/srb08abs205 ◽

2008 ◽

Vol 20 (9) ◽

pp. 5

Author(s):

P. Murthi ◽

N. Pathirage ◽

A. Borg ◽

S. Brennecke ◽

B. Kalionis

Keyword(s):

Fetal Growth ◽

Fetal Growth Restriction ◽

Homeobox Gene ◽

Growth Restriction ◽

Growth Potential ◽

Mrna Levels ◽

Developmental Abnormalities ◽

Clinical Criteria ◽

Targeted Mutation ◽

Clinically Significant

Fetal Growth Restriction (FGR) is a clinically significant pregnancy disorder in which the fetus fails to achieve its full growth potential in utero. Recently, we identified a novel homeobox gene TGIF, in the placenta using microarray expression profiling (1). Targeted mutation of tgif in mouse results in placental dysfunction (2). In this study, we have investigated TGIF expression levels in idiopathic FGR. FGR-affected placental samples were collected based on strict clinical criteria to ensure inclusion of cases at the severe end of the spectrum of the disease. TGIF mRNA expression was analysed in placentae obtained from pregnancies complicated by idiopathic FGR and gestation-matched control pregnancies (n = 25 each). Real-time PCR showed a significant increase in TGIF mRNA levels in FGR-affected placentae and gestation-matched controls [1.29 ± 0.06 FGR v. 0.78 ± 0.04 Control, P < 0.001]. western blotting using a TGIF polyclonal antibody revealed significantly increased levels of TGIF protein in term FGR-affected placentae compared with term controls [3970 ± 1101 (n = 10) v. 2323 ± 644 (n = 10), P < 0.05]. The spatial distribution of TGIF protein by immunohistochemistry revealed immunoreactive TGIF protein in residual cytotrophoblast cells, syncytiotrophoblast cells, microvascular endothelial cells and in stromal cells. We conclude that increased expression of homeobox gene TGIF may be a contributing factor to the developmental abnormalities seen in the FGR-affected placentae. (1) Murthi P, Hiden U, Rajaraman G, Kalionis B. Placenta May 29, [Epub ahead of print]. (2) Bartholin L, Melhuish TA et al. Dev Biol. 2008 May 2. [Epub ahead of print].

Download Full-text