J Korean Med Sci.  2009 Jan;24(Suppl 1):S176-S182. 10.3346/jkms.2009.24.S1.S176.

Hypoxia Induces Connective Tissue Growth Factor mRNA Expression

• Lee YK
• Kim EJ
• Lee JE
• Noh JW
• Kim YG

• KMID: 1778159
• DOI: http://doi.org/10.3346/jkms.2009.24.S1.S176

Abstract

Connective tissue growth factor (CTGF) is known to be a profibrotic growth factor, which mediate the fibrotic effect of transforming growth factor-beta(TGF-beta) and to stimulate cell proliferation and matrix production. CTGF has been shown to be hypoxiainducible in several cell types. Here we investigated the effect of hypoxia on CTGF gene expression in cultured mouse renal tubular cells (MTC). Quiescent cultures of MTC were exposed to hypoxia (1% O2) or normoxia in serum-free medium. The effects on hypoxia-induced CTGF expression were evaluated by Northern blot and real-time PCR. The roles of mitogen-activated protein kinase (MAPK) and TGF-beta were also determined using specific biochemical inhibitors. Exposure of quiescent tubular cells to hypoxia for 24 hr in a conditioned medium resulted in a significant increase TGF-beta. Hypoxia caused a significant increase in CTGF mRNA expression in MTC. Either JNK or ERK inhibitor did not block the hypoxia-induced stimulation of CTGF, whereas an inhibitor of p38 MAPK reduced the hypoxia-induced changes of CTGF. Although hypoxia stimulated TGF-betaproduction, neutralizing anti-TGF-beta1 antibody did not abolish the hypoxia-induced CTGF mRNA expression. The data suggest that hypoxia up-regulates CTGF gene expression, and that p38 MAPK plays a role in hypoxic-stimulation of CTGF. We also demonstrated that hypoxia induces CTGF mRNA expression via a TGF-beta1-independent mechanism.

Keyword

Cell Hypoxia; Connective Tissue Growth Factor; Transforming Growth Factor Beta 1; Mitogen-activated Protein Kinase

MeSH Terms

Animals
*Anoxia
Connective Tissue Growth Factor/*metabolism
Culture Media, Conditioned/metabolism
Enzyme-Linked Immunosorbent Assay
*Gene Expression Regulation
Kidney/metabolism
Kidney Tubules/cytology
MAP Kinase Signaling System
Mice
Models, Biological
RNA, Messenger/*metabolism
Reverse Transcriptase Polymerase Chain Reaction
Time Factors
Transforming Growth Factor beta/metabolism

Figure

• 

  Fig. 1 Time course of CTGF mRNA expression in response to hypoxia. MTC were isolated, cultured until 80% confluent, and exposed to 1% oxygen for 0-10 hr. CTGF transcripts were detected by Northern blot analysis; a representative blot is shown along with ethidium bromide stained 28S and 18S rRNA for loading. Densitometric analysis for CTGF mRNA levels is shown normalized to 18S rRNA level. Data are representative of three separate experiments. *p<0.05 compared with 0 hr sample.

• 

  Fig. 2 Effects of MAPK inhibitors on hypoxia-induced CTGF expression. Quiescent cells were pretreated for 30 min either with PD-098059 (PD, 50 µM/L), SB-203580 (SB, 20 µM/L), or JNK inhibitor (20 µM/L) followed by exposure to hypoxia or normoxia for 4 hr. At the end of incubations, total RNA was prepared from the cells and subjected to and RT-PCR. *p<0.05 compared with hypoxia.

• 

  Fig. 3 TGF-β1 stimulation of CTGF in normoxic condition. TGF-β1 induces CTGF mRNA in cultured MTC. Tubular cells were serum starved for 24 hr before being treated with 10 ng/mL TGF-β1. Cells were harvested at various time points and RNA was extracted. Northern hybridization for CTGF mRNA levels were performed and normalized against endogenous 18S ribosomal RNA. *p<0.05 compared with 0 hr sample.

• 

  Fig. 4 Effect of hypoxia on levels of TGF-β1 in MTC. MTC were isolated, cultured until 80% confluent, and exposed to 1% oxygen for 0-24 hr. Conditioned medium was collected at the end of hypoxia. TGF-β level in the cell medium was measured by a TGF-β ELISA kit. *p<0.05 compared with 0 hr sample.

• 

  Fig. 5 Effects of anti-TGF-β1 antibody on CTGF activity exposed to hypoxia. MTC were exposed to hypoxia in the presence of 2 µg/mL of anti-TGF-β1 antibody for 30 min, followed by exposure to hypoxia for 0-8 hr. Relative CTGF levels were calculated as a percentage of the relevant control values from arbitrary densitometry units. NS, not significant.

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