Sonic hedgehog signaling regulates Gli3 processing, mesenchymal proliferation, and differentiation during mouse lung organogenesis

Lack of Sonic hedgehog (Shh) signaling, mediated by the Gli proteins, leads to severe pulmonary hypoplasia. However, the precise role of Gli genes in lung development is not well established. We show Shh signaling prevents Gli3 proteolysis to generate its repressor forms (Gli3R) in the developing murine lung. In Shh(-/-) or cyclopamine-treated wild-type (WT) lung, we found that Gli3R level is elevated, and this upregulation appears to contribute to defects in proliferation and differentiation observed in the Shh(-/-) mesenchyme, where Gli3 is normally expressed. In agreement, we found Shh(-/-);Gli3(-/-) lungs exhibit enhanced growth potential. Vasculogenesis is also enhanced; in contrast, bronchial myogenesis remains absent in Shh(-/-);Gli3(-/-) compared with Shh(-/-) lungs. Genes upregulated in Shh(-/-);Gli3(-/-) relative to Shh(-/-) lung include Wnt2 and, surprisingly, Foxf1 whose expression has been reported to be Shh-dependent. Cyclins D1, D2, and D3 antibody labelings also reveal distinct expression patterns in the normal and mutant lungs. We found significant repression of Tbx2 and Tbx3, both linked to inhibition of cellular senescence, in Shh(-/-) and partial derepression in Shh(-/-); Gli3(-/-) lungs, while Tbx4 and Tbx5 expressions are less affected in the mutants. Our findings shed light on the role of Shh signaling on Gli3 processing in lung growth and differentiation by regulating several critical genes.     

Structural Basis for the Different Stability and Activity between the Cdk5 Complexes with p35 and p39 Activators

Cyclin-dependent kinase 5 (Cdk5) is a brain-specific membrane-bound protein kinase that is activated by binding to the p35 or p39 activator. Previous studies have focused on p35-Cdk5, and little is known regarding p39-Cdk5. The lack of functional understanding of p39-Cdk5 is due, in part, to the labile property of p39-Cdk5, which dissociates and loses kinase activity in nonionic detergent conditions. Here we investigated the structural basis for the instability of p39-Cdk5. p39 and p35 contain N-terminal p10 regions and C-terminal Cdk5 activation domains (AD). Although p35 and p39 show higher homology in the C-terminal AD than the N-terminal region, the difference in stability is derived from the C-terminal AD. Based on the crystal structures of the p25 (p35 C-terminal region including AD)-Cdk5 complex, we simulated the three-dimensional structure of the p39 AD-Cdk5 complex and found differences in the hydrogen bond network between Cdk5 and its activators. Three amino acids of p35, Asp-259, Asn-266, and Ser-270, which are involved in hydrogen bond formation with Cdk5, are changed to Gln, Gln, and Pro in p39. Because these three amino acids in p39 do not participate in hydrogen bond formation, we predicted that the number of hydrogen bonds between p39 and Cdk5 was reduced compared with p35 and Cdk5. Using substitution mutants, we experimentally validated that the difference in the hydrogen bond network contributes to the different properties between Cdk5 and its activators.     

New Structural Insights into Phosphorylation-free Mechanism for Full Cyclin-dependent Kinase (CDK)-Cyclin Activity and Substrate Recognition

Pho85 is a versatile cyclin-dependent kinase (CDK) found in budding yeast that regulates a myriad of eukaryotic cellular functions in concert with 10 cyclins (called Pcls). Unlike cell cycle CDKs that require phosphorylation of a serine/threonine residue by a CDK-activating kinase (CAK) for full activation, Pho85 requires no phosphorylation despite the presence of an equivalent residue. The Pho85-Pcl10 complex is a key regulator of glycogen metabolism by phosphorylating the substrate Gsy2, the predominant, nutritionally regulated form of glycogen synthase. Here we report the crystal structures of Pho85-Pcl10 and its complex with the ATP analog, ATPγS. The structure solidified the mechanism for bypassing CDK phosphorylation to achieve full catalytic activity. An aspartate residue, invariant in all Pcls, acts as a surrogate for the phosphoryl adduct of the phosphorylated, fully activated CDK2, the prototypic cell cycle CDK, complexed with cyclin A. Unlike the canonical recognition motif, SPX(K/R), of phosphorylation sites of substrates of several cell cycle CDKs, the motif in the Gys2 substrate of Pho85-Pcl10 is SPXX. CDK5, an important signal transducer in neural development and the closest known functional homolog of Pho85, does not require phosphorylation either, and we found that in its crystal structure complexed with p25 cyclin a water/hydroxide molecule remarkably plays a similar role to the phosphoryl or aspartate group. Comparison between Pho85-Pcl10, phosphorylated CDK2-cyclin A, and CDK5-p25 complexes reveals the convergent structural characteristics necessary for full kinase activity and the variations in the substrate recognition mechanism.     

Systemic administration of high-molecular weight hyaluronan stimulates wound healing in genetically diabetic mice

Hyaluronic acid (HA), an essential component of the extracellular matrix, is an efficient space filler that maintains hydration, serves as a substrate for assembly of proteoglycans and is involved in wound healing. Although numerous pieces of evidence demonstrate beneficial effects in promoting wound healing in diabetes, a systemic approach has never been tested. We used an incisional wound healing model in genetically diabetic mice to test the effects of systemic injection of HA. Diabetic (n = 56) and normoglycemic (n = 56) mice were subjected to incision and randomized (8 groups of 7 animals each) to receive HA at different doses, 7.5, 15 and 30 mg/kg/i.p., or vehicle (0.9% NaCl solution) for 12 days. At the end of the experiment animals were sacrificed and skin wounds were excised for histological, biochemical and molecular analysis. Histology revealed that the most effective dose to improve wound repair and angiogenesis in diabetic mice was 30 mg/kg. Furthermore HA injection (30 mg/kg) improved the altered healing pattern in diabetic animals, increased skin remodeling proteins TGF-β and transglutaminase-II and restored the altered expression of cyclin B1/Cdc2 complex. Evaluation of skin from diabetic animals injected with HA revealed also an increase in HA content, suggesting that systemic injection may be able to restore the reduced intracellular HA pool of diabetic mice. Finally HA markedly improved skin mechanical properties. These promising results, if confirmed in a clinical setting, may improve the care and management of diabetic patients.     

Cyclin D1 Is a Bona Fide Target Gene of NFATc1 and Is Sufficient in the Mediation of Injury-induced Vascular Wall Remodeling

Platelet-derived growth factor BB induced cyclin D1 expression in a time- and nuclear factor of activated T cells (NFAT)-dependent manner in human aortic smooth muscle cells (HASMCs), and blockade of NFATs prevented HASMC DNA synthesis and their cell cycle progression from G₁ to S phase. Selective inhibition of NFATc1 by its small interfering RNA also blocked HASMC proliferation and migration. Characterization of the cyclin D1 promoter revealed the presence of several NFAT binding sites, and the site at nucleotide −1333 was found to be sufficient in mediating platelet-derived growth factor BB-induced cyclin D1 promoter-luciferase reporter gene activity. In addition to its role in cell cycle progression, cyclin D1 mediated HASMC migration in an NFATc1-dependent manner. Balloon injury-induced cyclin D1-CDK4 activity requires NFAT activation, and adenovirus-mediated transduction of cyclin D1 was found to be sufficient to overcome the blockade effect of NFATs by VIVIT on balloon injury-induced vascular wall remodeling events, including smooth muscle cell migration from the medial to luminal region, their proliferation in the intimal region, and neointima formation. Together, these results provide more mechanistic evidence for the role of NFATs, particularly NFATc1, in the regulation of HASMC proliferation and migration as well as vascular wall remodeling. NFATc1 could be a potential therapeutic target against the renarrowing of artery after angioplasty.     

Novel Function and Intracellular Localization of Methionine Adenosyltransferase 2β Splicing Variants

Human methionine adenosyltransferase 2β (MAT2β) encodes for two major splicing variants, V1 and V2, which are differentially expressed in normal tissues. Both variants are induced in human liver cancer and positively regulate growth. The aim of this work was to identify interacting proteins of V1 and V2. His-tagged V1 and V2 were overexpressed in Rosetta pLysS cells, purified, and used in a pulldown assay to identify interacting proteins from human colon cancer cell line RKO cell lysates. The eluted lysates were subjected to Western blot and in solution proteomic analyses. HuR, an mRNA-binding protein known to stabilize the mRNA of several cyclins, was identified to interact with V1 and V2. Immunoprecipitation and Western blotting confirmed their interaction in both liver and colon cancer cells. These variant proteins are located in both nucleus and cytoplasm in liver and colon cancer cells and, when overexpressed, increased the cytoplasmic HuR content. This led to increased expression of cyclin D1 and cyclin A, known targets of HuR. When endogenous expression of V1 or V2 is reduced by small interference RNA, cytoplasmic HuR content fell and the expression of these HuR target genes also decreased. Knockdown of cyclin D1 or cyclin A blunted, whereas knockdown of HuR largely prevented, the ability of V1 or V2 overexpression to induce growth. In conclusion, MAT2β variants reside mostly in the nucleus and regulate HuR subcellular content to affect cell proliferation.     

Cdc6 protein activates p27KIP1-bound Cdk2 protein only after the bound p27 protein undergoes C-terminal phosphorylation

In mammalian cells Cdk2 activity during the G(1)-S transition is mainly controlled by p27(KIP1). Although the amount and subcellular localization of p27 influence Cdk2 activity, how Cdk2 activity is regulated during this phase transition still remains virtually unknown. Here we report an entirely new mechanism for this regulation. Cdc6 the AAA+ ATPase, known to assemble prereplicative complexes on chromosomal replication origins and activate p21(CIP1)-bound Cdk2, also activated p27-bound Cdk2 in its ATPase and cyclin binding motif-dependent manner but only after the p27 bound to the Cdk2 was phosphorylated at the C terminus. ROCK, which mediates a signal for cell anchorage to the extracellular matrix and activates the mTORC1 cascade as well as controls cytoskeleton assembly, was partly responsible for C-terminal phosphorylation of the p27. In vitro reconstitution demonstrated ROCK (Rho-associated kinase)-mediated phosphorylation of Cdk2-bound p27 at the C terminus and subsequent activation of the Cdk2 by Cdc6.     

Cyclin K-containing kinase complexes maintain self-renewal in murine embryonic stem cells

Protein phosphorylation plays an important role in the regulation of self-renewal and differentiation of embryonic stem cells. However, the responsible intracellular kinases are not well characterized. Here, we discovered that cyclin K protein was highly expressed in pluripotent embryonic stem cells but low in their differentiated derivatives or tissue-specific stem cells. Upon cell differentiation, the level of cyclin K protein was decreased. Furthermore, knockdown of cyclin K led to cell differentiation, which could be rescued by an expression construct resistant to RNA interference. Surprisingly, cyclin K did not interact with CDK9 protein in cells as thought previously. Instead, it associated with CrkRS (also known as CDK12) and CDC2L5 (also known as CDK13). Similar to cyclin K, both CDK12 and CDK13 proteins were highly expressed in murine embryonic stem cells and were decreased upon cell differentiation. Importantly, knockdown of either kinase resulted in differentiation. Thus, our studies have uncovered two novel protein kinase complexes that maintain self-renewal in embryonic stem cells.