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. 2006 Aug;17(8):3534-42.
doi: 10.1091/mbc.e05-11-1082. Epub 2006 May 24.

LRRC4, a putative tumor suppressor gene, requires a functional leucine-rich repeat cassette domain to inhibit proliferation of glioma cells in vitro by modulating the extracellular signal-regulated kinase/protein kinase B/nuclear factor-kappaB pathway

Minghua Wu  1 , Chen Huang, Kai Gan, He Huang, Qiong Chen, Jue Ouyang, Yunlian Tang, Xiaoling Li, Yixin Yang, Houde Zhou, Yanhong Zhou, Zhaoyang Zeng, Lan Xiao, Dan Li, Ke Tang, Shourong Shen, Guiyuan Li
Affiliations

LRRC4, a putative tumor suppressor gene, requires a functional leucine-rich repeat cassette domain to inhibit proliferation of glioma cells in vitro by modulating the extracellular signal-regulated kinase/protein kinase B/nuclear factor-kappaB pathway

Minghua Wu et al. Mol Biol Cell. 2006 Aug.

Abstract

We have previously reported that the LRRC4 gene, which contains a conserved leucine-rich repeat (LRR) cassette and an immunoglobulin (Ig) IgC2 domain, is associated with glioma suppression both in vitro and in vivo. The present study provides evidence that the conspicuous absence of LRRC4 in high-grade gliomas directly contributes to the increasing tumor grade. The loss of LRRC4 in U251 cells is caused by the loss of homozygosity at chromosome 7q32-ter. It was also found that LRRC4 requires a functional LRR cassette domain to suppress U251 cell proliferation. In the LRR cassette domain, the third LRR motif of the core LRR is found to be indispensable for the function of LRRC4. The inhibitory effect of LRRC4 is accompanied by a decrease in the expression of pERK, pAkt, pNF-kappaBp65, signal transducer and activator of transcription protein-3 (STAT3), and mutant p53, and an increase in the expression of c-Jun NH2-terminal kinase (JNK)2 and p-c-Jun, suggesting that LRRC4 plays a major role in suppressing U251 cell proliferation by regulating the extracellular signal-regulated kinase (ERK)/Akt/NF-kappaBp65, STAT3, and JNK2/c-Jun pathways. In conclusion, LRRC4 may act as a novel candidate of tumor suppressor gene. Therefore, the loss of LRRC4 function may be an important event in the progression of gliomas.

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Figures

Figure 1.
Figure 1.
Analysis of LRRC4 expression in gliomas and glioblastoma cell lines. (A) Northern blotting analysis of LRRC4 in glioblastoma cell lines and gliomas (top). Relative levels of RNA loading are shown as methylene blue staining of 28s RNA and GAPDH (bottom). (B) RT-PCR analysis of LRRC4 expression in gliomas, primary culture glioma cells, and glioblastoma cell lines. RNA from gliomas and cell lines was amplified for 25 and 35 cycles, respectively, using GAPDH (bottom) and LRRC4 (top) primers. Water was used as the negative control, and fetal brain cDNA and pcDNA3.1(+)-LRRC4 plasmid were used as the positive control templates. Lanes 1–4, primary culture tumor cells derived from grade II–III glioma; and lane 5, primary culture tumor cells derived from grade IV glioblastoma. (C) Schematic view of LRRC4 gene. The gene is composed of two exons. Exon 2 contains the entire ORF of LRRC4. The start and the end of the coding sequence are marked by ATG and TGA, respectively. (D) ORF of LRRC4 was amplified from genomic DNA of glioblastoma cell lines (marker −2-kb DNA ladder). A 528-base pair PCR product from GAPDH was used for the normalization of genomic DNA levels.
Figure 2.
Figure 2.
(A) Schematic illustration of the various domains of the LRRC4 protein by the SMART software. LRRNT and LRRCT indicate that the cysteine-rich regions flanking the core LRRs; Tm, transmembrane region. (B) Restriction enzyme analysis of pcDNA3.1(+)LRRC4/FLAG mutants. Plasmids were digested with BamHI and EcoRI and analyzed by agarose gel electrophoresis. Wild-type (wt) LRRC4 was used as a control. (C) Constructs of pcDNA3.1(+)/LRRC4-FLAG mutants were transfected into U251 cells, and the expression of mutant proteins was detected by immunoblotting with anti-M2/FLAG antibodies.
Figure 3.
Figure 3.
The third LRR motif of the core LRRs is indispensable for LRRC4 to inhibit U251 cell proliferation. (A) Comparison of the proliferation potential between the mock transfected U251 cells and U251 cells transfected with wild-type LRRC4 or selected LRRC4 deletion mutants by MTT assay. (B) The soft agar assay for U251 cells stably expressing wild-type LRRC4 or its mutants. (C) The cell cycle assay for U251 cells stably expressing wild-type LRRC4 or its mutants. All the experiments were repeated three times. *p < 0.01, compared with the mock-transfected control cells.
Figure 4.
Figure 4.
Analysis of the phosphorylation status of ERK2, PKB/Akt and NFκBp65 in the mock- and wild-type LRRC4-transfected U251 cells. (A) Western blotting assay showing phospho- and total protein levels of ERK2, Akt, and NFκBp65. (B) Quantitative analysis of the phospho- or total protein level change of ERK, Akt, and NF-κB by scanning densitometry. (C) Western blotting assay showing phospho- and total protein levels of ERK2, Akt, and NFκBp65 in the mock- and wild-type LRRC4-transfected U251 cells treated with PD98059, LY294002, or PMA. (D) Effect of exogenous Akt on pERK and pAkt in LRRC4/U251 cells.
Figure 5.
Figure 5.
Western blotting assay showing the change of the ERK/Akt/NF-κB, JNK2/c-Jun, STAT3 signaling pathways in the mock-, wild-type LRRC4, or its mutant-transfected U251 cells. (A) Protein levels of phospho-ERK2, Akt, and NFκBp65. (B) Protein levels of JNK2/p-c-Jun and mutant p53. (C) Protein level of STAT3. (D) Protein levels of cyclinD1.
Figure 6.
Figure 6.
Apoptosis assay of LRRC4 in the mock- and LRRC4-transfected U251cells. (A) Flow cytometry histogram displaying apoptosis apex in front of G1 phase. (B) AO/EB dual staining showing necrosis cells and apoptosis cells. (C) Western blotting image showing caspase-8 and caspase-3 at the protein level in the mock- and LRRC4-transfected U251 cells (+/− serum). β-actin is used as the loading control. (D) Real-time PCR products displaying caspase-8 and caspase-3 mRNA in the mock- and LRRC4-transfected U251 cells.
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