Regulation of insulin-like growth factor binding protein-5, four and a half lim-2, and a disintegrin and metalloprotease-9 expression in osteoblasts

Abstract

The roles of insulin-like growth factors (IGFs) in regulating growth and their modulation by six IGF binding proteins (IGFBP) are well established. IGFBP-5, the most abundant IGFBP stored in bone, is an important regulator of bone formation via IGF-dependent and -independent mechanisms. Two new proteins, four and a half lim (FHL)-2, a transcription modulator that interacts with IGFBP-5, and a disintegrin and metalloprotease (ADAM)-9, an IGFBP-5 protease, have been identified as potential regulators of IGFBP-5 action in bone. We tested the hypothesis that agents which modulate bone formation by regulating IGFBP-5 expression would also regulate FHL-2 and ADAM-9 expression in a coordinated manner. We evaluated the expression of IGFBP-5, FHL-2, and ADAM-9 by real-time reverse transcriptase (RT)-PCR during differentiation of mouse bone marrow stromal cells into osteoblasts and in response to treatment with bone formation modulators in the LSaOS human osteosarcoma cell line. IGFBP-5 and FHL-2 increased 4.3-and 3.0-fold (P ≤ 0.01), respectively, during osteoblast differentiation. Dexamethasone (Dex), an inhibitor of bone formation, decreased IGFBP-5 and FHL-2 and increased ADAM-9 in LSaOS cells (P ≤ 0.05). Bone morphogenic protein (BMP)-7, a stimulator of bone formation, increased IGFBP-5 and decreased ADAM-9 (P < 0.01). To determine if BMP-7 would eliminate Dex inhibition of IGFBP-5, cells were treated with Dex + BMP-7. The BMP-7-induced increase in IGFBP-5 was reduced, but not eliminated, in the presence of Dex (P ≤ 0.01), indicating that BMP-7 and Dex may regulate IGFBP-5 via different mechanisms. Transforming growth factor (TGF)-β, a stimulator of bone formation, increased IGFBP-5 and FHL-2 expression (P ≤ 0.01). IGF-I and TNF-α decreased expression of ADAM-9 (P < 0.05). In conclusion, our findings are consistent with the hypothesis that FHL-2 and ADAM-9 are important modulators of IGFBP-5 actions and are, in part, regulated in a coordinated manner in bone.

FHL2 expression in peritumoural fibroblasts correlates with lymphatic metastasis in sporadic but not in HNPCC-associated colon cancer

“…28 In contrast, FHL2 expression in colon cancer L Gullotti et al significant correlation of TGF-b1 expression and stromal FHL2 staining was not found in HNPCC-associated cancers, indicating that TGF-1 signalling from microsatellite-instable colon cancer cells may be defective. A histological difference between sporadic and HNPCC-associated cancers is the presence of dense lymphocytic infiltrates in the tumour micromilieu.…”

“…9 It has been shown that wounded tissue is comparable in several aspects to the tumour stroma as both are characterised by activated, a-SMA-expressing myofibroblasts and deposition of provisional matrix and basal membrane molecules. 11 As previous studies 19,28 described an induction of FHL2 expression by TGF-b1, this study considered both markers in the systematic analysis of colorectal tumour tissue and tumour stroma in vitro and in vivo.…”

Disruption of insulin-like growth factor-I expression in type IIαI collagen-expressing cells reduces bone length and width in mice

“…Quantitative real-time RT-PCR analysis was used to determine the expression levels of IGF-I, Cre, Col2␣1, GH receptor (GHR), IGF-I receptor (IGF-IR), parathyroid hormone (PTH)-related protein (PTHrP), Indian hedgehog (Ihh), PTH/PTHrP receptor (PTH1R), distal-less homeobox (Dlx)3, Dlx5, SRY-box containing gene (Sox)9, IGFBP-3 and -5, and PPIA (peptidylprolyl isomerase A; endogenous control) as previously described (13). PPIA was chosen as an internal control based on the determination that its expression is not different between different tissues or control and conditional mutant mice (data not shown).…”

“…Briefly, Stratagene Brilliant SYBRGreen Master Mix (Stratagene, La Jolla, CA) and a 7000 ABI Prism sequence detection system (Applied Biosystems, Foster City, CA) were used to determine gene expression. Primers were validated as previously described (13), and primer sequences are located in Table 1. Delta cycle threshold (Ct) values were determined (Ct value for gene of interest minus Ct value for control gene), and comparisons of the Ct values were used for relative quantification of gene expression (8).…”

Importin α-importin β complex mediated nuclear translocation of insulin-like growth factor binding protein-5

“…The endogenous IGFBP-5 is found in the nuclei of vascular smooth muscle cell culture and mouse embryonic cartilage cells and the N-domain of IGFBP-5 possess transactivation activity when fused to the DNA-binding domain of Gal4. FHL2 (four-and-a-half LIM domain 2), a transcriptional regulator, was found to interact with IGFBP-5 [21,22]. And also IGFBP-5 interacts with nuclear vitamin D receptor (VDR) to prevent retinoid X receptor (RXR): VDR heterodimerization [23].…”