A juxtamembrane basolateral targeting motif regulates signaling through a TGF-β pathway receptor in Drosophila

In polarized epithelial cells, receptor–ligand interactions can be restricted by different spatial distributions of the 2 interacting components, giving rise to an underappreciated layer of regulatory complexity. We explored whether such regulation occurs in the Drosophila wing disc, an epithelial tissue featuring the TGF-β family member Decapentaplegic (Dpp) as a morphogen controlling growth and patterning. Dpp protein has been observed in an extracellular gradient within the columnar cell layer of the disc, but also uniformly in the disc lumen, leading to the question of how graded signaling is achieved in the face of 2 distinctly localized ligand pools. We find the Dpp Type II receptor Punt, but not the Type I receptor Tkv, is enriched at the basolateral membrane and depleted at the junctions and apical surface. Wit, a second Type II receptor, shows a markedly different behavior, with the protein detected on all membrane regions but enriched at the apical side. Mutational studies identified a short juxtamembrane sequence required for basolateral restriction of Punt in both wing discs and mammalian Madin-Darby canine kidney (MDCK) cells. This basolateral targeting (BLT) determinant can dominantly confer basolateral localization on an otherwise apical receptor. Rescue of punt mutants with transgenes altered in the targeting motif showed that flies expressing apicalized Punt due to the lack of a functional BLT displayed developmental defects, female sterility, and significant lethality. We also show that apicalized Punt does not produce an ectopic signal, indicating that the apical pool of Dpp is not a significant signaling source even when presented with Punt. Instead, we find that basolateral presentation of Punt is required for optimal signaling. Finally, we present evidence that the BLT acts through polarized sorting machinery that differs between types of epithelia. This suggests a code whereby each epithelial cell type may differentially traffic common receptors to enable distinctive responses to spatially localized pools of extracellular ligands.

Receptor-ligand interactions can be restricted by different spatial distributions of the two interacting components, giving rise to an underappreciated layer of regulatory complexity. This study reveals that an evolutionarily conserved mechanism for polarized delivery of a transmembrane receptor (the Dpp Type II receptor Punt) supports robust morphogen signaling in a model epithelial tissue, the Drosophila wing disc.     

Macrophage migration inhibitory factor regulates joint capsule fibrosis by promoting TGF-β1 production in fibroblasts

Joint capsule fibrosis caused by excessive inflammation results in post-traumatic joint contracture (PTJC). Transforming growth factor (TGF)-β1 plays a key role in PTJC by regulating fibroblast functions, however, cytokine-induced TGF-β1 expression in specific cell types remains poorly characterized. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in inflammation- and fibrosis-associated pathophysiology. In this study, we investigated whether MIF can facilitate TGF-β1 production from fibroblasts and regulate joint capsule fibrosis following PTJC. Our data demonstrated that MIF and TGF-β1 significantly increased in fibroblasts of injured rat posterior joint capsules. Treatment the lesion sites with MIF inhibitor 4-Iodo-6-phenylpyrimidine (4-IPP) reduced TGF-β1 production and relieved joint capsule inflammation and fibrosis. In vitro, MIF facilitated TGF-β1 expression in primary joint capsule fibroblasts by activating mitogen-activated protein kinase (MAPK) (P38, ERK) signaling through coupling with membrane surface receptor CD74, which in turn affected fibroblast functions and promoted MIF production. Our results reveal a novel function of trauma-induced MIF in the occurrence and development of joint capsule fibrosis. Further investigation of the underlying mechanism may provide potential therapeutic targets for PTJC.     

TGF-β type I receptor Alk5 regulates tooth initiation and mandible patterning in a type II receptor-independent manner

TGF-β superfamily members signal through a heteromeric receptor complex to regulate craniofacial development. TGF-β type II receptor appears to bind only TGF-β, whereas TGF-β type I receptor (ALK5) also binds to ligands in addition to TGF-β. Our previous work has shown that conditional inactivation of Tgfbr2 in the neural crest cells of mice leads to severe craniofacial bone defects. In this study, we examine and compare the defects of TGF-β type II receptor (Wnt1-Cre;Tgfbr2^fl/fl) and TGF-β type I receptor/Alk5 (Wnt1-Cre;Alk5^fl/fl) conditional knockout mice. Loss of Alk5 in the neural crest tissue resulted in phenotypes not seen in the Tgfbr2 mutant, including delayed tooth initiation and development, defects in early mandible patterning and altered expression of key patterning genes including Msx1, Bmp4, Bmp2, Pax9, Alx4, Lhx6/7 and Gsc. Alk5 controls the survival of CNC cells by regulating expression of Gsc and other genes in the proximal aboral region of the developing mandible. We conclude that ALK5 regulates tooth initiation and early mandible patterning through a pathway independent of Tgfbr2. There is an intrinsic requirement for Alk5 signal in regulating the fate of CNC cells during tooth and mandible development.     

Osteoblastic Responses to TGF-β during Bone Remodeling

Bone remodeling depends on the spatial and temporal coupling of bone formation by osteoblasts and bone resorption by osteoclasts; however, the molecular basis of these inductive interactions is unknown. We have previously shown that osteoblastic overexpression of TGF-β2 in transgenic mice deregulates bone remodeling and leads to an age-dependent loss of bone mass that resembles high-turnover osteoporosis in humans. This phenotype implicates TGF-β2 as a physiological regulator of bone remodeling and raises the question of how this single secreted factor regulates the functions of osteoblasts and osteoclasts and coordinates their opposing activities in vivo. To gain insight into the physiological role of TGF-β in bone remodeling, we have now characterized the responses of osteoblasts to TGF-β in these transgenic mice. We took advantage of the ability of alendronate to specifically inhibit bone resorption, the lack of osteoclast activity in c-fos^−/− mice, and a new transgenic mouse line that expresses a dominant-negative form of the type II TGF-β receptor in osteoblasts. Our results show that TGF-β directly increases the steady-state rate of osteoblastic differentiation from osteoprogenitor cell to terminally differentiated osteocyte and thereby increases the final density of osteocytes embedded within bone matrix. Mice overexpressing TGF-β2 also have increased rates of bone matrix formation; however, this activity does not result from a direct effect of TGF-β on osteoblasts, but is more likely a homeostatic response to the increase in bone resorption caused by TGF-β. Lastly, we find that osteoclastic activity contributes to the TGF-β–induced increase in osteoblast differentiation at sites of bone resorption. These results suggest that TGF-β is a physiological regulator of osteoblast differentiation and acts as a central component of the coupling of bone formation to resorption during bone remodeling.     

Casein kinase 2 promotes the TGF-β-induced activation of α-tubulin acetyltransferase 1 in fibroblasts cultured on a soft matrix

Cell signals for growth factors depend on the mechanical properties of the extracellular matrix (ECM) surrounding the cells. Microtubule acetylation is involved in the transforming growth factor (TGF)-β-induced myofibroblast differentiation in the soft ECM. However, the mechanism of activation of α-tubulin acetyltransferase 1 (α-TAT1), a major α-tubulin acetyltransferase, in the soft ECM is not well defined. Here, we found that casein kinase 2 (CK2) is required for the TGF-β-induced activation of α-TAT1 that promotes microtubule acetylation in the soft matrix. Genetic mutation and pharmacological inhibition of CK2 catalytic activity specifically reduced microtubule acetylation in the cells cultured on a soft matrix rather than those cultured on a stiff matrix. Immunoprecipitation analysis showed that CK2α, a catalytic subunit of CK2, directly bound to the C-terminal domain of α-TAT1, and this interaction was more prominent in the cells cultured on the soft matrix. Moreover, the substitution of alanine with serine, the 236th amino acid located at the C-terminus, which contains the CK2-binding site of α-TAT1, sig-nificantly abrogated the TGF-β-induced microtubule acetylation in the soft matrix, indicating that the successful binding of CK2 and the C-terminus of α-TAT1 led to the phosphorylation of serine at the 236th position of amino acids in α-TAT1 and regulation of its catalytic activity. Taken together, our findings provide novel insights into the molecular mechanisms underlying the TGF-β-induced activation of α-TAT1 in a soft matrix.     

The histone demethylase Kdm6b regulates the maturation and cytotoxicity of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes

Intestinal intraepithelial lymphocytes (IELs) are distributed along the length of the intestine and are considered the frontline of immune surveillance. The precise molecular mechanisms, especially epigenetic regulation, of their development and function are poorly understood. The trimethylation of histone 3 at lysine 27 (H3K27Me3) is a kind of histone modifications and associated with gene repression. Kdm6b is an epigenetic enzyme responsible for the demethylation of H3K27Me3 and thus promotes gene expression. Here we identified Kdm6b as an important intracellular regulator of small intestinal IELs. Mice genetically deficient for Kdm6b showed greatly reduced numbers of TCRαβ⁺CD8αα⁺ IELs. In the absence of Kdm6b, TCRαβ⁺CD8αα⁺ IELs exhibited increased apoptosis, disturbed maturation and a compromised capability to lyse target cells. Both IL-15 and Kdm6b-mediated demethylation of histone 3 at lysine 27 are responsible for the maturation of TCRαβ⁺CD8αα⁺ IELs through upregulating the expression of Gzmb and Fasl. In addition, Kdm6b also regulates the expression of the gut-homing molecule CCR9 by controlling H3K27Me3 level at its promoter. However, Kdm6b is dispensable for the reactivity of thymic precursors of TCRαβ⁺CD8αα⁺ IELs (IELPs) to IL-15 and TGF-β. In conclusion, we showed that Kdm6b plays critical roles in the maturation and cytotoxic function of small intestinal TCRαβ⁺CD8αα⁺ IELs.Subject terms: Epigenetics, Gene regulation, Immunological disorders, T cells     

TDAG51 induces renal interstitial fibrosis through modulation of TGF-β receptor 1 in chronic kidney disease

Chronic kidney disease (CKD) is characterized by the gradual loss of renal function and is a major public health concern. Risk factors for CKD include hypertension and proteinuria, both of which are associated with endoplasmic reticulum (ER) stress. ER stress-induced TDAG51 protein expression is increased at an early time point in mice with CKD. Based on these findings, wild-type and TDAG51 knock-out (TDKO) mice were used in an angiotensin II/deoxycorticosterone acetate/salt model of CKD. Both wild-type and TDKO mice developed hypertension, increased proteinuria and albuminuria, glomerular injury, and tubular damage. However, TDKO mice were protected from apoptosis and renal interstitial fibrosis. Human proximal tubular cells were used to demonstrate that TDAG51 expression induces apoptosis through a CHOP-dependent mechanism. Further, a mouse model of intrinsic acute kidney injury demonstrated that CHOP is required for ER stress-mediated apoptosis. Renal fibroblasts were used to demonstrate that TGF-β induces collagen production through an IRE1-dependent mechanism; cells treated with a TGF-β receptor 1 inhibitor prevented XBP1 splicing, a downstream consequence of IRE1 activation. Interestingly, TDKO mice express significantly less TGF-β receptor 1, thus, preventing TGF-β-mediated XBP1 splicing. In conclusion, TDAG51 induces apoptosis in the kidney through a CHOP-dependent mechanism, while contributing to renal interstitial fibrosis through a TGF-β-IRE1-XBP1 pathway.Subject terms: Endoplasmic reticulum, Apoptosis, End-stage renal disease, Preclinical research, Chronic inflammation     

Numb-PRRL promotes TGF-β1- and EGF-induced epithelial-to-mesenchymal transition in pancreatic cancer

Isoform-specific functions of Numb in the development of cancers, especially in the initiation of epithelial-to-mesenchymal transition (EMT) remains controversial. We study the specific function of Numb-PRRL isoform in activated EMT of pancreatic ductal adenocarcinoma (PC), which is distinguished from our previous studies that only focused on the total Numb protein. Numb-PRRL isoform was specifically overexpressed and silenced in PC cells combining with TGF-β1 and EGF stimulus. We systematically explored the potential effect of Numb-PRRL in the activated EMT of PC in vitro and in vivo. The total Numb protein was overexpressed in the normal pancreatic duct and well-differentiated PC by IHC. However, Numb-PRRS isoform but not Numb-PRRL showed dominant expression in PC tissues. Numb-PRRL overexpression promoted TGF-β1-induced EMT in PANC-1 and Miapaca-2 cells. TGF-β1-induced EMT-like cell morphology, cell invasion, and migration were enhanced in Numb-PRRL overexpressing groups following the increase of N-cadherin, Vimentin, Smad2/3, Snail1, Snail2, and cleaved-Notch1 and the decrease of E-cadherin. Numb-PRRL overexpression activated TGFβ1-Smad2/3-Snail1 signaling was significantly reversed by the Notch1 inhibitor RO4929097. Conversely, Numb-PRRL silencing inhibited EGF-induced EMT in AsPC-1 and BxPC-3 cells following the activation of EGFR-ERK/MAPK signaling via phosphorylating EGFR at tyrosine 1045. In vivo, Numb-PRRL overexpression or silencing promoted or inhibited subcutaneous tumor size and distant liver metastases via regulating EMT and Snail signaling, respectively. Numb-PRRL promotes TGF-β1- and EGF-induced EMT in PC by regulating TGF-β1-Smad2/3-Snail and EGF-induced EGFR-ERK/MAPK signaling.Subject terms: Tumour-suppressor proteins, Cell invasion     

TGF-β receptor expression on human keratinocytes: A 150 kDa GPI-anchored TGF-β1 binding protein forms a heteromeric complex with type I and type II receptors

Keratinocytes play a critical role in re-epithelialization during wound healing, and alterations in keratinocyte proliferation and function are associated with the development of various skin diseases. Although it is well documented that TGF-β has profound effects on keratinocyte growth and function, there is a paucity of information on the types, isoform specificity and complex formation of TGF-β receptors on keratinocytes. Here, we report that in addition to the types I, II, and III TGF-β receptors, early passage adult and neonatal human keratinocytes display a cell surface glycosylphosphatidylinositol (GPI)-anchored 150 kDa TGF-β1 binding protein. The identities of the four proteins were confirmed on the basis of their affinity for TGF-β isoforms, immunoprecipitation with specific anti-receptor antibodies, sensitivity to phosphatidylinositol specific phospholipase C and dithiothreitol, and 2-dimensional electrophoresis. Interestingly, the antitype I TGF-β receptor antibody immunoprecipitated not only the type I receptor, but also the type II receptor and the 150 kDa component, suggesting that the 150 kDa component form heteromeric complexes with the signalling receptors. In addition, two-dimensional (nonreducing/reducing) electrophoresis confirmed the occurrence of a heterotrimeric complex consisting of the 150 kDa TGF-β1 binding protein, the type II receptor, and the type I receptor. This technique also demonstrated the occurrence of types I and II heterodimers and type I homodimers of TGF-β receptors on keratinocytes, supporting the heterotetrameric model of TGF-β signalling proposed using mutant cells and cells transfected to overexpress these receptors. The keratinocytes responded to TGF-β by markedly downregulating all four TGF-β binding proteins and by potently inhibiting DNA synthesis. The demonstration that the 150 kDa GPI-anchored TGF-β1 binding protein forms a heteromeric complex with the TGF-β signalling receptors suggests that this GPI-anchored protein may modify TGF-β signalling in human keratinocytes. J. Cell. Biochem. 70:573–586, 1998. © 1998 Wiley-Liss, Inc.     

Lin28a attenuates TGF-β-induced renal fibrosis

Lin28a has diverse functions including regulation of cancer, reprogramming and regeneration, but whether it promotes injury or is a protective reaction to renal injury is unknown. We studied how Lin28a acts in unilateral ureteral obstruction (UUO)-induced renal fibrosis following unilateral ureteral obstruction, in a mouse model. We further defined the role of Lin28a in transforming growth factor (TGF)-signaling pathways in renal fibrosis through in vitro study using human tubular epithelium-like HK-2 cells. In the mouse unilateral ureteral obstruction model, obstruction markedly decreased the expression of Lin28a, increased the expression of renal fibrotic markers such as type I collagen, α-SMA, vimentin and fibronectin. In TGF-β-stimulated HK-2 cells, the expression of Lin28a was reduced and the expression of renal fibrotic markers such as type I collagen, α-SMA, vimentin and fibronectin was increased. Adenovirus-mediated overexpression of Lin28a inhibited the expression of TGF-β-stimulated type I collagen, α-SMA, vimentin and fibronectin. Lin28a inhibited TGF-β-stimulated SMAD3 activity, via inhibition of SMAD3 phos-phorylation, but not the MAPK pathway ERK, JNK or p38. Lin28a attenuates renal fibrosis in obstructive nephropathy, making its mechanism a possible therapeutic target for chronic kidney disease.     

A large domain common to sperm receptors (Zp2 and Zp3) and TGF-β type III receptor

A new family of mosaic proteins is defined by sequence analysis. The family is characterized by a 260 residue domain common to proteins of apparently diverse function and tissue specificity; sperm receptors Zp2 and Zp3, betaglycan (also called TGF-β type III receptor), uromodulin, as well as the major zymogen granule membrane protein (GP-2). The location of the common domain is similar with respect to putative transmembrane regions. The results lead to the hypothesis that this type of domain has a common tertiary structure and that there is a functional similarity in the recognition mechanism of the sperm receptor system and the TGF-β receptor complex.     

Association of v-ErbA with Smad4 Disrupts TGF-β Signaling

Disruption of the transforming growth factor-β (TGF-β) pathway is observed in the majority of cancers. To further understand TGF-β pathway inactivation in cancer, we stably expressed the v-ErbA oncoprotein in TGF-β responsive cells. v-ErbA participates in erythroleukemic transformation of cells induced by the avian erythroblastosis virus (AEV). Here we demonstrate that expression of v-ErbA was sufficient to antagonize TGF-β–induced cell growth inhibition and that dysregulation of TGF-β signaling required that v-ErbA associate with the Smad4 which sequesters Smad4 in the cytoplasm. We also show that AEV-transformed erythroleukemia cells were resistant to TGF-β–induced growth inhibition and that TGF-β sensitivity could be recovered by reducing v-ErbA expression. Our results reveal a novel mechanism for oncogenic disruption of TGF-β signaling and provide a mechanistic explanation of v-ErbA activity in AEV-induced erythroleukemia.     

Cross Talk between β1 and αV Integrins: β1 Affects β3 mRNA Stability

There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of beta(1)-null GD25 cells ectopically expressing the beta(1)A integrin subunit, we provide evidence for the existence of a cross talk between beta(1) and alpha(V) integrins that affects the ratio of alpha(V)beta(3) and alpha(V)beta(5) integrin cell surface levels. In particular, we demonstrate that a down-regulation of alpha(V)beta(3) and an up-regulation of alpha(V)beta(5) occur as a consequence of beta(1)A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms beta(1)B and beta(1)D, as well as two beta(1) cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (beta(1)TR) or only its "variable" region (beta(1)COM), we show that the effects of beta(1) over alpha(V) integrins take place irrespective of the type of beta(1) isoform, but require the presence of the "common" region of the beta(1) cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby beta(1) integrins exert their trans-acting functions, we have found that the down-regulation of alpha(V)beta(3) is due to a decreased beta(3) subunit mRNA stability, whereas the up-regulation of alpha(V)beta(5) is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability.     

Modulation of gene expression in the preimplantation mouse embryo by TGF-α and TGF-β

The effect of growth factors on regulating gene expression in the preimplantation mouse embryo was examined, since results of previous experiments revealed a stimulatory effect of exogenously-added growth factors on preimplantation development in vitro. Treatment of early cavitating blastocysts with either 250 pM TGF-α or TGF-β results in changes in the pattern of total protein synthesis as assessed by high-resolution two-dimensional gel electrophoresis. In some cases, the synthesis of a particular polypeptide is either up- or downregulated by each growth factor, whereas in other instances the synthesis of a polypeptide is modulated by one but not the other growth factor. Use of the mRNA differential display method permitted the identification of genes whose expression is either up- or downregulated by these growth factors. Treatment of mouse blastocysts with either TGF-α or TGF-β results in the increased expression of the b subunit of the F₀ ATPase. TGF-β also stimulates the expression of the DNA polymerase α. TGF-α treatment results in the increase in expression of a gene homologous to the human HEPG2 cDNA, as well as in a decrease in expression of fibronectin. © 1995 Wiley-Liss, Inc.     

Conditional Expression of Smad7 in Pancreatic β Cells Disrupts TGF-β Signaling and Induces Reversible Diabetes Mellitus

Identification of signaling pathways that maintain and promote adult pancreatic islet functions will accelerate our understanding of organogenesis and improve strategies for treating diseases like diabetes mellitus. Previous work has implicated transforming growth factor-β (TGF-β) signaling as an important regulator of pancreatic islet development, but has not established whether this signaling pathway is required for essential islet functions in the adult pancreas. Here we describe a conditional system for expressing Smad7, a potent inhibitor of TGF-β signaling, to identify distinct roles for this pathway in adult and embryonic β cells. Smad7 expression in Pdx1 ⁺ embryonic pancreas cells resulted in striking embryonic β cell hypoplasia and neonatal lethality. Conditional expression of Smad7 in adult Pdx1 ⁺ cells reduced detectable β cell expression of MafA, menin, and other factors that regulate β cell function. Reduced pancreatic insulin content and hypoinsulinemia produced overt diabetes that was fully reversed upon resumption of islet TGF-β signaling. Thus, our studies reveal that TGF-β signaling is crucial for establishing and maintaining defining features of mature pancreatic β cells.