Insulin-like growth factor binding protein-3 is upregulated in LPS-treated THP-1 cells.
BACKGROUND
Lipopolysaccharide (LPS) is a potent activator of human monocytic cells. We have determined that LPS stimulation of the human monocytic cell line, THP-1, results in an increased apoptotic rate. We hypothesized that cDNA expression array analysis could be used to identify target genes involved in the regulation of this process.


METHODS
THP-1 cells (1 x 10(6)/mL) were stimulated with LPS (1 microg/mL) or vehicle control. Apoptosis was measured at 0, 24, 48, 72 and 96 h using propidium iodide staining and flow cytometry to determine the percentage of cells with hypodiploid DNA. At 16 h, the Atlas Human cDNA expression array system, containing probes for 205 genes related to apoptosis, was used to survey and quantify transcript expression. The experiment was performed in duplicate and the membranes were normalized to cytoplasmic beta-actin. Standard Western blotting was performed on the conditioned medium to correlate secreted protein expression with RNA expression. Pretreatment with insulin-like growth factor I (IGF-I) was performed to determine whether the effects of insulin-like growth factor binding protein-3 (IGFBP-3) on apoptosis were IGF-dependent.


RESULTS
LPS stimulation of THP-1 cells resulted in a greater than 2-fold increase in the rate of apoptosis when compared to vehicle control. When the cDNA expression arrays were compared, there was a 500-fold increase in the expression of the IGFBP-3 transcript in the LPS-stimulated cells. Western blotting of culture medium verified an approximately 2-fold increase in secreted IGFBP-3. Pretreatment with IGF-I did not prevent the increase in apoptosis seen with LPS stimulation.


CONCLUSIONS
THP-1 cell apoptosis is increased in response to LPS stimulation and is associated with a significant induction of IGFBP-3 mRNA and protein. IGFBP-3, which reportedly promotes apoptosis and modulates the bioavailability of the pro-survival insulin-like growth factor 1, may serve to regulate apoptosis in monocytic cells in an IGF-independent manner. These data further support the investigation of the role of the IGF axis in programmed cell death of immune cells.
Agnese
Doreen M
DM
Division of Surgical Sciences, Department of Surgery, UMDNJ-Robert Wood Johnson Medical School, One Robert Wood Johnson Place, New Brunswick, NJ 08903, USA.
Calvano
Jacqueline E
JE
Hahm
Sae J
SJ
Calvano
Steve E
SE
Lowry
Stephen F
SF
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002461:Cell Line, Transformed; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000096:biosynthesis* / Q000235:genetics; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008070:Lipopolysaccharides; D008213:Lymphocyte Activation; D009000:Monocytes; D020411:Oligonucleotide Array Sequence Analysis; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology*
D018972:Insulin-Like Growth Factor Binding Protein 3; D008070:Lipopolysaccharides; D007334:Insulin-Like Growth Factor I