Hypoxia-inducible factor-2 is a novel regulator of aberrant CXCL12 expression in multiple myeloma plasma cells

Abstract

Background: Multiple myeloma is an incurable malignancy of bone marrow plasma cells. Progression of multiple myeloma is accompanied by an increase in bone marrow angiogenesis. Studies from our laboratory suggest a role for the CXCL12 chemokine in this process, with circulating levels of CXCL12 correlating with bone marrow angiogenesis in patients with multiple myeloma. While the mechanisms responsible for aberrant plasma cell expression of CXCL12 remain to be determined, studies in other systems suggest a role for hypoxia and hypoxia-inducible transcription factors.

Design and methods: The expression of hypoxia-inducible factor protein was examined in patients' bone marrow biopsy specimens using immunohistochemistry. The hypoxic regulation of CXCL12 was examined in multiple myeloma plasma cell lines using polymerase chain reaction and western blotting. The role of hypoxia-inducible factors-1 and -2 in the regulation of CXCL12 expression was examined using over-expression and short hairpin RNA knockdown constructs, electrophoretic mobility shift assays and chromatin immunoprecipitation. The contribution of CXCL12 to hypoxia-induced angiogenesis was examined in vivo using a subcutaneous murine model of neovascularization.

Results: Strong hypoxia-inducible factor-2 protein expression was detected in CD138(+) multiple myeloma plasma cells in patients' biopsy specimens. Prolonged exposure to hypoxia strongly up-regulated CXCL12 expression in multiple myeloma plasma cells and hypoxia-inducible factor-2 was found to play a key role in this response. Promoter analyses revealed increased hypoxia-inducible factor-2 binding to the CXCL12 promoter under hypoxic conditions. Over-expression of hypoxia-inducible factor in multiple myeloma plasma cells strongly induced in vivo angiogenesis, and administration of a CXCL12 antagonist decreased hypoxia-inducible factor-induced angiogenesis.

Conclusions: Hypoxia-inducible factor-2 is a newly identified regulator of CXCL12 expression in multiple myeloma plasma cells and a major contributor to multiple myeloma plasma cell-induced angiogenesis. Targeting the hypoxic niche, and more specifically hypoxia-inducible factor-2, may represent a viable strategy to inhibit angiogenesis in multiple myeloma and progression of this disease.

Figure 1.

Expression of CXCL12, HIF-1α and HIF-2α in patients’ trephine specimens. Bone marrow trephine sections from (A) MGUS and (B) MM patients at diagnosis were co-stained with CXCL12 (a and b, pink), HIF-1α (c and d, pink) or HIF-2α (e and f, pink) and CD138 (all sections, brown). Original magnifications x40 (a, c and e) and x200 (b, d and f); colors corrected after acquisition with Adobe Photoshop.

Figure 2.

Hypoxic regulation of CXCL12 expression in LP-1 cells. (A) Levels of CXCL12 mRNA expression were assessed in LP-1 cells following 6, 24 and 48 h of normoxic (white bars) or hypoxic (black bars) culture. Columns, mean (n=3); bars, SEM. *P<0.05, **P<0.001, compared to normoxia. (B) Levels of CXCL12 protein were measured in LP-1 conditioned medium following 72 h of normoxic or hypoxic culture. Columns, mean (n=3); bars, SEM. *P<0.001, compared to normoxia. (C) The hypoxic induction of CXCL12 (black bars) mRNA, and HIF-1α (dotted line) and HIF-2α (dashed line) protein expression was examined in LP-1 cells over 72 h. Columns and dashed lines, mean (n=3); bars, SEM.

Figure 3.

Stable over-expression of HIF-1α and HIF-2α in LP-1 cells. (A) LP-1 cells were engineered to stably over-express HIF-1α or HIF-2α and up-regulation of HIF protein confirmed by western immunoblotting. (B) Levels of CXCL12 mRNA expression were examined in the HIF over-expressing LP-1 cell lines. Columns, mean (n=3); bars, SEM. **P<0.001, compared to vector control. (C) Levels of CXCL12 protein were measured in conditioned media from the HIF over-expressing cell lines. Columns, mean (n=3); bars, SEM. *P<0.05, **P<0.001, compared to vector control.

Figure 4.

Stable knockdown of HIF-1α and HIF-2α in LP-1 cells. (A) RNA interference was used to knock down endogenous HIF-1α or HIF-2α expression in LP-1 cells, and down-regulation of HIF protein confirmed by western immunoblotting. (B) Levels of CXCL12 mRNA expression were measured in the HIF knockdowns in response to normoxic (white bars) or hypoxic (black bars) culture. Columns, mean (n=3); bars, SEM. *P<0.05, **P<0.001, compared to vector control. (C) Levels of CXCL12 protein were measured in conditioned media from the HIF knockdowns following 72 h of normoxic (white bars) or hypoxic (black bars) culture. Columns, mean (n=3); bars, SEM. *P<0.05, compared to vector control.

Figure 5.

Hypoxia induces HIF-2α binding to the CXCL12 promoter in LP-1 cells. (A) Human CXCL12 locus containing two HIF binding site (HBS) sequences and transcriptional start site. Hypoxic induction of CXCL12 is primarily mediated via HBS1. (B) LP-1 cells were transiently transfected with a luciferase plasmid containing the proximal CXCL12 promoter (top, pGL3b-CXCL12). Twenty-four hours post-transfection, cells were cultured for 48 h under normoxic (white bars) or hypoxic (black bars) conditions, and luciferase assays performed. Columns, mean (n=3); bars, SEM. *P<0.05, compared to normoxia. (C) The pGL3b-CXCL12 construct (top) was transiently transfected into the HIF-over-expressing LP-1 cell lines and luciferase assays performed. Columns, mean (n=3); bars, SEM. *P<0.05, compared to vector control. (D) LP-1 cells were cultured under normoxic (N, lane 1) or hypoxic (H, lane 2) conditions for 48 h and DNA binding activity to HBS1 examined by electromobility shift assay. To determine the contribution of HIF-2 to the hypoxia-inducible complex formation, extracts were pre-incubated with HIF-2α antibody (lanes 3 and 4). (E) Chromatin immunoprecipitation was performed on LP-1 cells cultured under normoxic (white bars) or hypoxic (black bars) conditions for 48 h, and the level of HIF-2α binding to HBS1 of the CXCL12 promoter assessed by PCR. Columns, mean; bars, SEM. *P<0.05, compared to normoxia.

Figure 6.

HIF-induced CXCL12 stimulates in vivo angiogenesis. (A) CXCL12-, HIF-1α- or HIF-2α-over-expressing LP-1 cells were injected subcutaneously in a Matrigel plug into mice (n=12/group), and half of the mice were administered the CXCR4 antagonist, T140. After 2 weeks, mice were euthanized and macroscopic photographs were taken of the implants. (B) The hemoglobin content of implants from untreated (white bars) and T140-treated (black bars) mice were measured and normalized to cell burden as described in the Design and Methods sections. Columns, mean (n=6/group); bars, SEM. *P<0.05, compared to vector control.