Autocrine BMP4 signaling involves effect of cholesterol myristate on proliferation of mesenchymal stem cells.

We recently identified that cholesterol myristate in traditional Chinese medicine (TCM) is the active compound that increases proliferation of mesenchymal stem cell (MSCs). The present study is further to determine what signal pathway involves in effect of cholesterol myristate. Reverse transcription-PCR, Western blot and ELISA analysis show that cholesterol myristate increases the release of bone morphogenetic protein 4 (BMP4) from MSCs and the expression in the intracellular levels of BMP4 in a time- and dose dependent manner. However, structurally related steroids such as cholesterol and cholesten presented in TCM, both lack of the myristate, did not affect the secretion and expression of BMP4 on MSCs. These finds suggest that myristate is essential for the effects of cholesterol myristate. Furthermore, cholesterol myristate significantly increase BMPRIB levels of MSCs and the number of BMPRIB positive cells in a time- and dose dependent manner, but not BMPR IA or BMPR II. Our results indicate that action of cholesterol myristate may activate the BMP4-BMPRIB autocrine. Moreover, a blocking antibody against BMP4 or the BMP4 antagonist, noggin, partially reduced the effects of cholesterol myristate on MSCs proliferation. Thus, this study is to provide evidence that autocrine BMP4 signaling involves effect of cholesterol myristate on MSCs proliferation.

Bone morphogenetic protein 4 inhibits TGF-beta2 stimulation of extracellular matrix proteins in optic nerve head cells: role of gremlin in ECM modulation.

The characteristic cupping of the optic nerve head (ONH) in glaucoma is associated with elevated TGF-beta2 and increased synthesis and deposition of extracellular matrix (ECM) proteins. In addition to TGF-beta2, the human ONH also expresses bone morphogenetic proteins (BMPs) and BMP receptors, which are members of the TGF-beta superfamily. We examined the potential effects of BMP4 and the BMP antagonist gremlin on TGF-beta2 induction of ECM proteins in ONH cells. BMP-4 dose dependently inhibited TGF-beta2-induced fibronectin (FN) and PAI-1 expression in ONH astrocytes and lamina cribrosa (LC) cells and also reduced TGF-beta2 stimulation of collagen I, collagen VI, and elastin. Addition of gremlin blocked this BMP-4 response, increasing cellular and secreted FN as well as PAI-1 levels in both cell types. Gremlin was expressed in ONH tissues and ONH cells, and gremlin protein levels were significantly increased in the LC region of human glaucomatous ONH tissues. Interestingly, recombinant gremlin dose dependently increased ECM protein expression in cultured ONH astrocytes and LC cells. Gremlin stimulation of ECM required activation of TGF-beta receptor and R-Smad3. TGF-beta2 increased gremlin mRNA expression and protein levels in ONH cells. Inhibition of either the type I TGF-beta receptor or Smad3 phosphorylation blocked TGF-beta2-induced gremlin expression. In conclusion, BMP4 blocked the TGF-beta2 induction of ECM proteins in ONH cells. The BMP antagonist gremlin reversed this inhibition, allowing TGF-beta2 stimulation of ECM synthesis. Increased expression of gremlin in the glaucomatous ONH may further exacerbate TGF-beta2 effects on ONH ECM metabolism by inhibiting BMP-4 antagonism of TGF-beta2 signaling. Modulation of the ECM via gremlin provides a novel therapeutic target for glaucoma.