CaMKII inhibition in human primary and pluripotent stem cell-derived chondrocytes modulates effects of TGFß and BMP through SMAD signaling.

OBJECTIVE: Upregulation of calcium/calmodulin-dependent kinase II (CaMKII) is implicated in the pathogenesis of osteoarthritis (OA) and reactivation of articular cartilage hypertrophy. However, direct inhibition of CaMKII unexpectedly augmented symptoms of OA in animal models. The role of CaMKII in OA remains unclear and requires further investigation. METHODS: Analysis of CaMKII expression was performed in normal human and OA articular chondrocytes, and signaling mechanisms were assessed in articular, fetal and Pluripotent Stem Cell (PSC)-derived human chondrocytes using pharmacological (KN93), peptide (AC3-I) and small interfering RNA (siRNA) inhibitors of CaMKII. RESULTS: Expression levels of phospho-CaMKII (pCaMKII) were significantly and consistently increased in human OA specimens. BMP2/4 activated expression of pCaMKII as well as COLII and COLX in human adult articular chondrocytes, and also increased the levels and nuclear localization of SMADs1/5/8, while TGFß1 showed minimal or no activation of the chondrogenic program in adult chondrocytes. Targeted blockade of CaMKII with specific siRNAs decreased levels of pSMADs, COLII, COLX and proteoglycans in normal and OA adult articular chondrocytes in the presence of both BMP4 and TGFß1. Both human fetal and PSC-derived chondrocytes also demonstrated a decrease of chondrogenic differentiation in the presence of small molecule and peptide inhibitors of CaMKII. Furthermore, immunoprecipitation for SMADs1/5/8 or 2/3 followed by western blotting for pCaMKII showed direct interaction between SMADs and pCaMKII in primary chondrocytes. CONCLUSION: Current study demonstrates a direct role for CaMKII in TGF-ß and BMP-mediated responses in primary and PSC-derived chondrocytes. These findings have direct implications for tissue engineering of cartilage tissue from stem cells and therapeutic management of OA.

Antagonistic Smad transcription factors control the dauer/non-dauer switch in C. elegans.

The C. elegans daf-8 gene encodes an R-Smad that is expressed in a subset of head neurons, the intestine, gonadal distal tip cells and the excretory cell. We found that DAF-8, which inhibits the DAF-3 Co-Smad, is associated with DAF-3 and the DAF-14 Smad in vivo and in vitro. Overexpression of daf-8 conferred a dauer-defective phenotype and suppressed constitutive dauer formation in daf-8 and daf-14 mutants. In contrast to mammalian systems described thus far, active DAF-3 drives a feedback regulatory loop that represses transcription of daf-7 (a TGFbeta ligand) and daf-8 by directly binding to their regulatory regions. Hence, DAF-8 and DAF-3 are mutually antagonistic. The feedback repression may reinforce the developmental switch by allowing DAF-3 to freely activate dauer transcription in target tissues, unless sufficiently inhibited by DAF-8 and DAF-14. In the adult, DAF-8 downregulates lag-2 expression in the distal tip cells, thus promoting germ line meiosis. This function does not involve DAF-3, thereby avoiding the feedback loop that functions in the dauer switch.

Direct visualization of Smad1/5/8-mediated transcriptional activity identifies podocytes and collecting ducts as major targets of BMP signalling in healthy and diseased kidneys.

Bone morphogenetic protein 7 (BMP7) is a key determinant of renal response to injury, exhibiting strong protective as well as regenerative potential in a variety of experimental models. In vitro, beneficial effects of stimulation with BMP7 and other BMPs have been observed in many renal cell types. Still, it remains poorly understood which cells in the native kidney actually respond to BMPs in health and disease. Here, we report the use of BRE:gfp mice expressing green fluorescent protein (GFP) under the control of a pSmad1/5/8-specific BMP-responsive element (BRE) to directly visualize the spatiotemporal distribution of transcriptional activity downstream of canonical BMP signalling in healthy kidneys and in two distinct models of kidney disease. BRE-GFP signal coincided with expression of endogenous BMP target genes but, surprisingly, it was much more restricted than expected from the widespread distribution of pSmad1/5/8, a classical component of canonical BMP signal tranduction. BRE-GFP was mainly present in podocytes and collecting duct cells, and both glomerular and medullary BRE-GFP decreased following ischaemia-reperfusion injury as well as following unilateral ureteric obstruction, together with decreased BMP7, pSmad1/5/8 and BMP target gene expression. Remarkably, however, BRE-GFP was increased in injured proximal tubules in association with up-regulation of BMP receptors ALK2 and ALK3. Thus, native BMP transcriptional activity is much more restricted than previously suggested based on pSmad1/5/8 detection alone, and its response to injury varies according to cell type and nephron segment.

Essential role for Smad3 in angiotensin II-induced tubular epithelial-mesenchymal transition.

Angiotensin II (Ang II) is a key mediator of chronic kidney disease, in which epithelial-mesenchymal transition (EMT) is a critical process mediated by the TGFbeta/Smad signalling pathway. The present study examined the specific role of Smads in Ang II-induced EMT in vitro and in vivo. We found that Ang II signalled through the receptor of AT1, not AT2, to activate Smad2/3 and induce EMT in a normal rat tubular epithelial cell line (NRK52E). Activation of Smads by Ang II was attributed to degradation of an inhibitory Smad7, which was mediated by the AT1-Smurf2-dependent ubiquitin degradation mechanism because blockade of AT1 receptor or knockdown of Smurf2 inhibited Smad7 loss, thereby reducing Smad2/3 activation and EMT in response to Ang II. In contrast, over-expression of Smad7 inhibited Ang II-induced Smad2/3 activation and EMT in NRK52E cells and in a rat model of remnant kidney disease. Moreover, knockdown of Smad3, not Smad2, attenuated Ang II-induced EMT. In conclusion, Ang II activates Smad signalling to induce EMT, which is mediated by a loss of Smad7 through the AT1-Smurf2-dependent ubiquitin degradation pathway. Smad3, but not Smad2, may be a mediator of EMT, while Smad7 may play a protective role in EMT in response to Ang II.

Muscle-derived Myoglianin regulates Drosophila imaginal disc growth.

Organ growth and size are finely tuned by intrinsic and extrinsic signaling molecules. In Drosophila, the BMP family member Dpp is produced in a limited set of imaginal disc cells and functions as a classic morphogen to regulate pattern and growth by diffusing throughout imaginal discs. However, the role of TGFß/Activin-like ligands in disc growth control remains ill-defined. Here, we demonstrate that Myoglianin (Myo), an Activin family member, and a close homolog of mammalian Myostatin (Mstn), is a muscle-derived extrinsic factor that uses canonical dSmad2-mediated signaling to regulate wing size. We propose that Myo is a myokine that helps mediate an allometric relationship between muscles and their associated appendages.

Mechanisms of TGFbeta inhibition of LUNG endodermal morphogenesis: the role of TbetaRII, Smads, Nkx2.1 and Pten.

Transforming growth factor-beta is a multifunctional growth factor with roles in normal development and disease pathogenesis. One such role is in inhibition of lung branching morphogenesis, although the precise mechanism remains unknown. In an explant model, all three TGFbeta isoforms inhibited FGF10-induced morphogenesis of mesenchyme-free embryonic lung endoderm. Inhibition of budding by TGFbeta was partially abrogated in endodermal explants from Smad3(-/-) or conditional endodermal-specific Smad4(Delta/Delta) embryonic lungs. Endodermal explants from conditional TGFbeta receptor II knockout lungs were entirely refractive to TGFbeta-induced inhibition. Inhibition of morphogenesis was associated with dedifferentiation of endodermal cells as documented by a decrease in key transcriptional factor, NKX2.1 protein, and its downstream target, surfactant protein C (SpC). TGFbeta reduced the proliferation of wild-type endodermal cells within the explants as assessed by BrdU labeling. Gene expression analysis showed increased levels of mRNA for Pten, a key regulator of cell proliferation. Conditional, endodermal-specific deletion of Pten overcame TGFbeta's inhibitory effect on cell proliferation, but did not restore morphogenesis. Thus, the mechanisms by which TGFbeta inhibits FGF10-induced lung endodermal morphogenesis may entail both inhibition of cell proliferation, through increased Pten, as well as inhibition or interference with morphogenetic mediators such as Nkx2.1. Both of the latter are dependent on signaling through TbetaRII.

[BMP in the kidney: signaling, function and pathophysiological significance].

Bone morphogenetic proteins (BMPs) are multipotent signaling molecules that belong to the transforming growth factor-beta (TGF-beta) superfamily. Developmentally these proteins promote endochondral bone formation and are involved in the cascade of body patterning and morphogenesis. Moreover, BMPs play an important role in the pathophysiology of several diseases, including osteoporosis, arthritis, pulmonary hypertension, cerebrovascular diseases, cancer and kidney diseases. In this review, BMP signaling and regulation, the pathophysiological role of BMP in kidney diseases and potential therapeutic applications have been discussed.

Activin inhibits the human Pit-1 gene promoter through the p38 kinase pathway in a Smad-independent manner.

The pituitary transcription factor Pit-1 regulates hormonal production from the anterior pituitary gland. However, the mechanisms by which Pit-1 gene expression is regulated in humans are poorly understood. Activin, a member of the TGFbeta superfamily, acts as a negative regulator of cell growth and prolactin gene expression in lactotrope cells. In this study, we show that activin negatively regulates the human Pit-1 gene promoter. We defined a 117-bp element within the Pit-1 promoter that is sufficient to relay these inhibitory effects. We further investigated the signaling pathways that mediate activin-induced inhibition of Pit-1 gene promoter in pituitary lactotrope cells. We found that the activin effects on Pit-1 gene regulation are Smad independent and require the p38 MAPK pathway. Specifically, blocking p38 kinase activity reverses activin-mediated inhibition of the Pit-1 gene promoter. Together, our results highlight the p38 MAPK pathway as a key regulator of activin function in pituitary lactotrope cells and further emphasizes the critical role played by activin in regulating hormonal production in the pituitary gland.

Cellular and molecular basis for the regulation of inflammation by TGF-beta.

Transforming growth factor-beta (TGF-beta) has been shown to play an essential role in the suppression of inflammation, yet recent studies have revealed the positive roles of TGF-beta in inflammatory responses. For example, TGF-beta induces Foxp3-positive regulatory T cells (iTregs) in the presence of interleukin-2 (IL-2), while in the presence of IL-6, it induces pathogenic IL-17 producing Th17 cells. TGF-beta inhibits the proliferation of immune cells as well as cytokine production via Foxp3-dependent and -independent mechanisms. Little is known about molecular mechanisms involved in immune suppression via TGF-beta; however, Smad2/3 have been shown to play essential roles in Foxp3 induction as well as in IL-2 and IFN-gamma suppression, whereas Th17 differentiation is promoted via the Smad-independent pathway. Interaction between TGF-beta and other cytokine signaling is important in establishing the balance of immunity and tolerance.

Nodal signals pattern vertebrate embryos.

Vertebrate embryonic patterning requires several conserved inductive signals-including Nodal, Bmp, Wnt and Fgf signals. Nodal, which is a member of the transforming growth factor beta (TGFbeta) superfamily, activates a signal transduction pathway that is similar to that of other TGFbeta members. Nodal genes, which have been identified in numerous vertebrate species, are expressed in specific cell types and tissues during embryonic development. Nodal signal transduction has been shown to play a pivotal role in inducing and patterning mesoderm and endoderm, and in regulating neurogenesis and left-right axis asymmetry. Antagonists, which act at different steps in the Nodal signal transduction pathway, have been shown to tightly modulate the inductive activity of Nodal.

Identification of a Smad phosphatase.

Activation of Smad signaling pathways downstream of TGF-beta superfamily ligands via receptor-mediated Smad phosphorylation is well understood, but little is known about the phosphatases that turn off Smad activity. Now in Cell, Feng and colleagues (Lin, X., et al. (2006) Cell 125 , 915-928) report their discovery that PPM1A acts as a Smad phosphatase to terminate TGF-beta signaling.