Loss of murine Gfi1 causes neutropenia and induces osteoporosis depending on the pathogen load and systemic inflammation
Fig 3
Diminished osteoblast and osteoclast activity in nonSPF Gfi1-ko/ko mice.
(A) The Ob. and Oc. activity in Gfi1-ko/ko and Gfi1-wt/wt mice kept at nonSPF conditions was determined by measuring the plasma markers receptor activator of NF-kB ligand (Rankl), osteoprogerin (Opg), cross-linked carboxy-terminal telopeptide of type I collagen (CTX-I), and osteocalcin. Elevated Opg and diminished Rankl levels in Gfi1-ko/ko mutants suggest reduced osteoclast activity. Diminished bone resorption and formation in Gfi1-ko/ko mice is indicated by lowered CTX-I and osteocalcin levels, respectively. Error bars represent SD and statistical significance was calculated with t-test, * p ≤ 0.05 and ** p ≤ 0.01. (B) Basal expression of Gfi1 mRNA in different tissues shows highest values in spleen, bone marrow, and cortical bone. Non-haematopoietic tissues such as liver or cartilage and in vitro differentiated Oc. as well as Ob. demonstrated very low Gfi1 mRNA expression (n = 3 with 3 technical replicates/sample). Gfi1 expression was assessed with quantitative PCR (qPCR) and Gapdh was used as endogenous control. (C) All markers of Ob. proliferation and differentiation are diminished in Gfi1-ko/ko mutant lysates. Please note the low levels of osteoblast markers Ocn, Runx2, and Osx but normal expression of the osteocyte marker Dmp1. Values are shown as ratio of Gfi1-ko/ko vs. Gfi1-wt/wt mRNA expression (n = 3 with 3 technical replicates/sample). Normal expression is indicated with the dotted line at 1. Relative expression of bone marker genes was assessed by qPCR in full RNA preparations of cortical bone. Gapdh was used as endogenous control. (D) Immunohistology of the Ob. marker osteocalcin (Bglap) demonstrates abundant signals (red) in controls at the bone marrow to bone junction but low levels in Gfi1-ko/ko mutants. Please note abundant osteocalcin signals also in bone marrow cells. Immunostaining was performed on film supported cryosections. Counterstaining occurred with DAPI visualizing cell nuclei.