TGFβ is secreted as part of a latent complex that is targeted to the extracellular matrix. A variety of molecules, `TGFβ activators,' release TGFβ from its latent state. The unusual temporal discontinuity of TGFβ synthesis and action and the panoply of TGFβ effects contribute to the interest in TGF-β. However, the logical connections between TGFβ synthesis, storage and action are obscure. We consider the latent TGFβ complex as an extracellular sensor in which the TGFβ propeptide functions as the detector, latent-TGFβ-binding protein (LTBP) functions as the localizer, and TGF-β functions as the effector. Such a view provides a logical continuity for various aspects of TGFβ biology and allows us to appreciate TGFβ biology from a new perspective.

Productive interaction of a T lymphocyte with an antigen-presenting cell results in the clustering of the T-cell antigen receptor (TCR) and the recruitment of a large signalling complex to the site of cell-cell contact. Subsequent signal transduction resulting in cytokine gene expression requires the activation of one or more of the multiple isoenzymes of serine/threonine-specific protein kinase C (PKC). Among the several PKC isoenzymes expressed in T cells, PKC-theta is unique in being rapidly recruited to the site of TCR clustering. Here we show that PKC-theta is essential for TCR-mediated T-cell activation, but is dispensable during TCR-dependent thymocyte development. TCR-initiated NF-kappaB activation was absent from PKC-theta(-/-) mature T lymphocytes, but was intact in thymocytes. Activation of NF-kappaB by tumour-necrosis factor alpha and interleukin-1 was unaffected in the mutant mice. Although studies in T-cell lines had suggested that PKC-theta regulates activation of the JNK signalling pathway, induction of JNK was normal in T cells from mutant mice. These results indicate that PKC-theta functions in a unique pathway that links the TCR signalling complex to the activation of NF-kappaB in mature T lymphocytes.

Transforming growth factor-βs (TGF-β) are secreted as inactive complexes containing the TGF-β, the TGF-β propeptide, also called the latency-associated protein (LAP), and the latent TGF-β binding protein (LTBP). Extracellular activation of this complex is a critical but incompletely understood step in TGF-β regulation. We have investigated the role of LTBP in modulating TGF-β generation by the integrin αVβ6. We show that even though αvβ6 recognizes an RGD on LAP, LTBP-1 is required for αVβ6-mediated latent TGF-β activation. The domains of LTBP-1 necessary for activation include the TGF-β propeptide-binding domain and a basic amino acid sequence (hinge domain) with ECM targeting properties. Our results demonstrate an LTBP-1 isoform-specific function in αVβ6-mediated latent TGF-β activation; LTBP-3 is unable to substitute for LTBP-1 in this assay. The results reveal a functional role for LTBP-1 in latent TGF-β activation and suggest that activation of specific latent complexes is regulated by distinct mechanisms that may be determined by the LTBP isoform and its potential interaction with the matrix.

Diabetes is a pathological condition characterized by relative insulin deficiency, persistent hyperglycemia, and, consequently, diffuse micro-and macrovascular disease. One therapeutic strategy is to amplify insulin-secretion capacity by increasing the number of the insulin-producing β cells without triggering a generalized proliferative response. Here, we present the development of a small-molecule screening platform for the identification of molecules that increase β-cell replication. Using this platform, we identify a class of compounds [adenosine kinase inhibitors (ADK-Is)] that promote replication of primary β cells in three species (mouse, rat, and pig). Furthermore, the replication effect of ADK-Is is cell type-selective: treatment of islet cell cultures with ADK-Is increases replication of β cells but not that of α cells, PP cells, or fibroblasts. Short-term in vivo treatment with an ADK-I also increases β-cell replication but not exocrine cell or hepatocyte replication. Therefore, we propose ADK inhibition as a strategy for the treatment of diabetes.

Transforming growth factors-L L (TGFL L1, 2 and 3) are secreted in a complex with their propeptides (latency-associated peptide 1 (LAP1), 2 and 3). TGFL L signaling requires the dissociation of LAP and TGFL L, a process termed latent TGFL L activation. This process is a critical but incompletely understood step in the regulation of TGFL L function. In particular, the extent to which activation mechanisms differ among the three TGFL L isoforms is relatively unexplored. We show here that K K V L L 6 binds and activates latent TGFL L3. ß

Pharmacologic expansion of endogenous β cells is a promising therapeutic strategy for diabetes. To elucidate the molecular pathways that control β-cell growth we screened ∼2400 bioactive compounds for rat β-cell replication-modulating activity. Numerous hit compounds impaired or promoted rat β-cell replication, including CC-401, an advanced clinical candidate previously characterized as a c-Jun N-terminal kinase inhibitor. Surprisingly, CC-401 induced rodent (in vitro and in vivo) and human (in vitro) β-cell replication via dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) 1A and 1B inhibition. In contrast to rat β cells, which were broadly growth responsive to compound treatment, human β-cell replication was only consistently induced by DYRK1A/B inhibitors. This effect was enhanced by simultaneous glycogen synthase kinase-3β (GSK-3β) or activin A receptor type II-like kinase/transforming growth factor-β (ALK5/TGF-β) inhibition. Prior work emphasized DYRK1A/B inhibition-dependent activation of nuclear factor of activated T cells (NFAT) as the primary mechanism of human β-cell-replication induction. However, inhibition of NFAT activity had limited effect on CC-401-induced β-cell replication. Consequently, we investigated additional effects of CC-401-dependent DYRK1A/B inhibition. Indeed, CC-401 inhibited DYRK1A-dependent phosphorylation/stabilization of the β-cell-replication inhibitor p27Kip1. Additionally, CC-401 increased expression of numerous replication-promoting genes normally suppressed by the dimerization partner, RB-like, E2F and multivulval class B (DREAM) complex, which depends upon DYRK1A/B activity for integrity, including MYBL2 and FOXM1. In summary, we present a compendium of compounds as a valuable resource for manipulating the signaling pathways that control β-cell replication and leverage a DYRK1A/B inhibitor (CC-401) to expand our understanding of the molecular pathways that control β-cell growth.

Insulin initiates diverse hepatic metabolic responses, including gluconeogenic suppression and induction of glycogen synthesis and lipogenesis1,2. The liver possesses a rich sinusoidal capillary network with increased hypoxia and decreased gluconeogenesis in the perivenous zone3. Here, diverse vascular endothelial growth factor (VEGF) inhibitors improved glucose tolerance in normal or diabetic db/db mice, potentiating hepatic insulin signaling, decreasing gluconeogenic gene expression, increasing glycogen storage and suppressing hepatic glucose production (HGP). VEGF inhibition induced hepatic hypoxia via sinusoidal vascular regression and sensitized liver insulin signaling through hypoxia inducible factor-2α (HIF-2α) stabilization. Notably, liver-specific constitutive activation of HIF-2α, but not HIF-1α, was sufficient to augment hepatic insulin signaling via direct and indirect induction of insulin receptor substrate 2 (IRS2), an essential insulin receptor adaptor protein4–6. Further, liver IRS2 was both necessary and sufficient to mediate HIF-2α and VEGF inhibition effects on glucose tolerance and hepatic insulin signaling. These results demonstrate an unsuspected intersection between HIF-2α–mediated hypoxic signaling and hepatic insulin action via IRS2 induction, which can be co-opted by VEGF inhibitors to modulate glucose metabolism. These studies also indicate distinct roles in hepatic metabolism for HIF-1α, which promotes glycolysis7–9, versus HIF-2α, which suppresses gluconeogenesis, and suggest novel treatment approaches for type 2 diabetes mellitus.

The latent transforming growth factor-beta-binding protein-1 (LTBP-1) belongs to a family of extracellular glycoproteins that includes three additional isoforms (LTBP-2, -3, and -4) and the matrix proteins fibrillin-1 and -2. Originally described as a TGF-beta-masking protein, LTBP-1 is involved both in the sequestration of latent TGF-beta in the extracellular matrix and the regulation of its activation in the extracellular environment. Whereas the expression of LTBP-1 has been analyzed in normal and malignant cells and rodent and human tissues, little is known about LTBP-1 in embryonic development. To address this question, we used murine embryonic stem (ES) cells to analyze the appearance and role of LTBP-1 during ES cell differentiation. In vitro, ES cells aggregate to form embryoid bodies (EBs), which differentiate into multiple cell lineages. We analyzed LTBP-1 gene expression and LTBP-1 fiber appearance with respect to the emergence and distribution of cell types in differentiating EBs. LTBP-1 expression increased during the first 12 d in culture, appeared to remain constant between d 12 and 24, and declined thereafter. By immunostaining, fibrillar LTBP-1 was observed in those regions of the culture containing endothelial, smooth muscle, and epithelial cells. We found that inclusion of a polyclonal antibody to LTBP-1 during EB differentiation suppressed the expression of the endothelial specific genes ICAM-2 and von Willebrand factor and delayed the organization of differentiated endothelial cells into cord-like structures within the growing EBs. The same effect was observed when cultures were treated with either antibodies to TGF-beta or the latency associated peptide, which neutralize TGF-beta. Conversely, the organization of endothelial cells was enhanced by incubation with TGF-beta 1. These results suggest that during differentiation of ES cells LTBP-1 facilitates endothelial cell organization via a TGF-beta-dependent mechanism.