Durand et al. 10.1073/pnas.0706923105. |
Fig. 5. Gremlin inhibits the activity of recombinant BMP4. To verify that gremlin antagonizes the activity of BMP4, we used an in vitro culture system where the stromal cell line UG26.1B6 was induced with BMP4 to differentiate into osteoblasts (1, 2). UG26.1B6 cells were cultured at a density of 1-2 ´ 104 cells per cm2 in 10% FCS a-MEM medium supplemented with BMP4 (50 ng/ml) in the absence (Upper) or in the presence (Lower) of recombinant gremlin (250 ng/ml) for 14 days. Differentiation of UG26.1B6 cells was examined by the detection of alkaline phosphatase (ALP) activity, an early marker of osteogenic differentiation (1). The presence of gremlin during the culture period prevents the BMP4-induced differentiation of osteoblastic cells, because no ALP+ cells were found. The figure is representative of four independent experiments, each performed in duplicate.
2. Hsu DR, Economides AN, Wang X, Eimon PM, Harland RM (1998) Mol Cell 1:673-683.
Table 3. Genes commonly expressed by UG26.1B6 and UG26.3B5 cells
Categories of cell regulators | Gene products identified by transcriptional profiling |
Cyto/chemokines, receptors, angiogenic factors | M-CSF, SCF, IGF, IL1-RI, IL-10Rb, LIF-R, gp130, GM-CSFRa, SDF-1, lymphotactin, CXCL10, MLP-1, VEGFa-d, MIP-1g, b-NGF |
TGFb and TNF-related molecules | TGFb, TGFb2, BmprII, TGFbRI, Bmp4, Bmp11, TNFa, FasL, Thank, TNFRI, gremlin |
Developmental factors | Neuropilin 1, neuropilin 2, semaphorin 3C, Notch2 |
Adhesion | Integrin a5, integrin-av, integrin-b1, integrin-b5, VCAM-1 |
Signal transduction | c-jun, c-myc, en-1, en-2, jak-1, NF-kB, Smad1,2,4,6,7, Stat1 |
Apoptosis | Bcl-x, caspase 3, cox-1, cox-3, survivin, Bax-a, TRP53/p53, AIF, Apaf1, Bag1, SARP-1 |
Other | ADAM-10 (metalloprotease), TERT, TRF1, TR (telomerase-related) |
It is of particular interest to note that SCF, IGF, VEGF, TNFRI, TGFbRI, BMPRII, and Notch signaling genes, were previously identified by Hackney et al. (1) in fetal liver stroma. These combined data suggest that generally the same panels of molecules are expressed and possibly relevant in several developmental and anatomically distinct hematopoietic supportive microenvironments. It has yet to be determined what specific concentrations of these molecules, alone and in combination, affect HSC growth.
1. Hackney JA, Charbord P, Brunk BP, Stoeckert CH, Lemischka IR, Moore KA (2002) Proc Natl Acad Sci USA 99:13061-13066.
Table 4. Sequences of q-PCR and RT-PCR primers
Gene | Sequence | Size product, bp | |
Forward q-PCR | Reverse q-PCR | ||
b -NGF | gtgccgagcctccaatcc | ccggatgaacctccaagc | 139 |
MIP-1g | gcagtctgaaggcacagc | tggcacagacctggaacc | 191 |
Bmp4 | agcgtcccgccagccga | cggagctctgccgaggag | 148 |
Forward RT-PCR | Reverse RT-PCR | ||
b -NGF | gtgccgagcctccaatcc | ccggatgaacctccaagc | 139 |
p75 | ccctgcacggtgtgcgag | cggccggctgttggctcc | 372 |
TrkA | ggtaccagctctccaacactgagg | ccagaacgtccaggtaactcggtg | 204 |
MIP-1g | ggcccagatcacacatgc | accaagccatctctccag | 593 |
CCR1 | gctcatgcagcataggagg | catggccaggtccagttgc | 677 |
Bmp4 | cccagagaatgaggtgatctcc | tggcagtagaaggcctggtag | 569 |
Alk3 | gctccatggcactggtatg | cacaaccagccatcggatg | 382 |
Alk6 | gccagctggttccgagag | agccaagcccaggtctgc | 347 |
BmprII | gtgccagctggccaggca | ggcgccaccgcttaagag | 468 |
Smad1 | gtgctggttccgaagcac | ggcagtggaggcgccatc | 326 |
Smad4 | gcggcagtgtcaccggca | gctgtgggtccgcaatgg | 328 |
Smad5 | cagcctgtcgcctatgag | cactgcagaggcccgtgg | 556 |
b -actin | cctgaaccctaaggccaaccg | gctcatagctcttctccaggg | 397 |