Effect of bone morphogenetic protein-6 on macrophages

Abstract

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)-beta superfamily which regulates bone formation, haematopoiesis and development. While TGF-beta is known to be a negative regulator of the immune system, the effect of BMPs on the immune system is largely unknown. Herein, the effect of BMP-6 on the innate immune system was investigated using the murine macrophage cell line RAW 264.7. BMP-6 altered cellular morphology, inhibited cellular proliferation, increased the fraction of subG(1) phase cells, and decreased the fraction of cells in the S and G(2)M phases, without changing the percentage of apoptotic cells. In addition, BMP-6 induced expression of pro-inflammatory inducible nitric oxide synthase (iNOS) and the cytokine tumour necrosis factor (TNF)-alpha. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated the expression of all three known type II BMP receptors [BMP-RII, activin (Act)-RIIA and Act-RIIB] and two of the three known type I receptors [activin receptor-like kinase 2 (ALK2) and ALK3]. Over-expression as well as knock-down studies using short hairpin RNA (shRNA) demonstrated that BMP-RII, ALK2 and ALK3 are the functional BMP-6 receptors in macrophages. Finally, the effect of BMP-6 was confirmed in murine peritoneal macrophages and the THP-1 human monocyte cell line. Taken together, these results demonstrate that BMP-6 regulates the proliferation and gene expression profile of macrophages.

Figure 1

Effects of bone morphogenetic protein-6 (BMP-6) on RAW 264·7 cells. (a) BMP-6 dramatically altered the morphology of RAW 264·7 cells, similarly to lipopolysaccharide (LPS) treatment. (b) Cellular proliferation was determined by haemocytometer. Treatment with BMP-6 suppressed the proliferation of RAW 264·7 cells in a concentration-dependent manner (*P ≤ 0·05) (top panel). BMP-6 also inhibited cellular proliferation of primary murine peritoneal macrophages and the human monocyte/macrophage cell line THP-1 in a concentration-dependent manner (*P < 0·05) (bottom panel). Bars represent the average cell count ± standard deviation (SD). (c, d) Cell cycle analysis by flow cytometry showed an increased fraction of cells in the G₁ phase and a decreased fraction in the S and G₂/M phases after BMP-6 treatment (*P ≤ 0·05). Bars represent the average cell count ± SD. (e) RAW 264·7 cells were treated with 100 ng of BMP-6 for 24 and 48 hr and the apoptotic population was measured using fluorescence-activated cell sorting (FACS). PMφ, peritoneal macrophage φ.

Figure 2

Bone morphogenetic protein-6 (BMP-6) induced production of inducible nitric oxide synthase (iNOS) and tumour necrosis factor (TNF)-α. The expression of BMP receptors was increased in RAW 264·7 cells. (a) BMP response element (BRE) activity was increased by BMP treatment. Cells were transfected with the reporter construct BRE-luciferase (luc). As the concentration of BMP-6 increased, the levels of luciferase activity increased (*P ≤ 0·05). Bars represent the average cell count ± standard deviation (SD). (b) Effects of BMP-6 on the expression of iNOS and TNF-α in RAW 264·7 cells. Reverse transcription–polymerase chain reaction (RT-PCR) was carried out to determine the effect of BMP-6 on the expression of iNOS and TNF-α. The expression of iNOS and TNF-α increased with BMP-6 exposure in a dose-dependent manner. (c) In RT-PCR analysis for type I and type II BMP receptors, all BMP receptors with the exception of activin receptor-like kinase 6 (ALK6) were found to be highly expressed in RAW 264·7 cells (top panel). A similar expression pattern was found in primary murine peritoneal macrophages and the human monocyte/macrophage cell line THP-1 (bottom panel).

Figure 3

Functional type I bone morphogenetic protein (BMP) receptor in RAW 264·7 cells. (a) Cells were transfected with constitutively active activin receptor-like kinase 2 (ALK2) or ALK3 (ALK2-CA and ALK3-CA, respectively) along with BMP response element-luciferase (BRE-luc). Compared with the control (in which cells were transfected with the empty expression vector pcDAN3·1), there was a significant increase in luciferase activity level when cells were transfected with either ALK2-CA or ALK3-CA. (b) An experiment identical to that in (a) was carried out and the effect on cell count was determined 48 hr after transfection. Compared with the control (pcDAN3·1), cells transfected with either ALK2-CA or ALK3-CA demonstrated a significant decrease in cell count. (c) RAW 264·7 cells were transfected with a lentivirus-based short hairpin RNA (shRNA) construct, and expression of type I receptors by these cells was detected by RT-PCR. The mRNA level was decreased for clone 1 of ALK2 and clone 2 of ALK3. Con, control; #1, #2 and #3, clones 1, 2 and 3, respectively. (d) Effect of the shRNA construct on cell growth. The growth inhibitory effect of BMP-6 was abolished by shRNA. (e) Induction of inducible nitric oxide synthase (iNOS) and tumour necrosis factor (TNF)-α in shRNA-transfected cells. In ALK2- and shALK3-transfected cells, BMP-6 could not induce iNOS and TNF-α.

Figure 4

Functional type II bone morphogenetic protein (BMP) receptor in RAW 264·7 cells. (a) Cells were transfected with each of the three type II BMP receptors along with the reporter plasmid BMP response element-luciferase (BRE-luc). Following treatment with BMP-6, all three type II receptors increased the luciferase activity in transfected cells. However, cells transfected with BMP-RII demonstrated the highest level of luciferase activity. (b) Following transfection with each of the three type II BMP receptors, cells were treated with BMP-6 at 100 ng/ml. At the end of a 48-hr period, the effect on cell count was determined. As expected, only the proliferation of cells transfected with BMP-RII was inhibited with BMP-6. Cells transfected with the empty expression vector pcDAN3·1 and treated with BMP-6 were used as the control. To control for varying transfection efficiency, cells were co-transfected with the reporter construct CMV-LacZ. (c) RAW 264·7 cells were transfected with lentiviral constructs containing short hairpin RNA (shRNA) targeting each of the three BMP type II receptors. Three sequences were tested for each of the type II BMP receptors. The effect on the levels of expression of the target was determined by reverse transcription–polymerase chain reaction (RT-PCR). Con, control; #1, #2 and #3, clones 1, 2 and 3, respectively. (d) RAW 264·7 cells were transfected with lentivirus containing a shRNA sequence targeting each of the type II BMP receptors. Subsequently, cells were treated with 100 ng/ml of BMP-6 for 48 hr. The results demonstrated that BMP-6-induced growth inhibition was reversed only when the expression of BMP-RII was knocked down. (e) Induction of iNOS and TNF-α was investigated in shRNA transfected cells. ShBMP-RII completely blocked induction of inducible nitric oxide synthase (iNOS) and tumour necrosis factor (TNF)-α. However, the shRNA construct of activin (Act)-RIIa and Act-RIIb only partially blocked the induction of iNOS and TNF-α.