Disruption of the creb3l1 gene causes defects in caudal fin regeneration and patterning in zebrafish Danio rerio.

BACKGROUND: The gene cAMP-Responsive Element Binding protein 3-like-1 (CREB3L1) has been implicated in bone development in mice, with CREB3L1 knock-out mice exhibiting fragile bones, and in humans, with CREB3L1 mutations linked to osteogenesis imperfecta. However, the mechanism through which Creb3l1 regulates bone development is not fully understood. RESULTS: To probe the role of Creb3l1 in organismal physiology, we used CRISPR-Cas9 genome editing to generate a Danio rerio (zebrafish) model of Creb3l1 deficiency. In contrast to mammalian phenotypes, the Creb3l1 deficient fish do not display abnormalities in osteogenesis, except for a decrease in the bifurcation pattern of caudal fin. Both, skeletal morphology and overall bone density appear normal in the mutant fish. However, the regeneration of caudal fin postamputation is significantly affected, with decreased overall regenerate and mineralized bone area. Moreover, the mutant fish exhibit a severe patterning defect during regeneration, with a significant decrease in bifurcation complexity of the fin rays and distalization of the bifurcation sites. Analysis of genes implicated in bone development showed aberrant patterning of shha and ptch2 in Creb3l1 deficient fish, linking Creb3l1 with Sonic Hedgehog signaling during fin regeneration. CONCLUSIONS: Our results uncover a novel role for Creb3l1 in regulating tissue growth and patterning during regeneration.

Lack of Nuclear Localization of the Creb3l1 Transcription Factor Causes Defects in Caudal Fin Bifurcation in Zebrafish Danio rerio.

INTRODUCTION: The formation of normal bone and bone healing requires the cAMP-responsive element binding protein 3-like-1 (Creb3l1) transmembrane transcription factor, as deletion of the murine CREB3L1 results in osteopenic animals with limited capacity to repair bone after a fracture. Creb3l1 undergoes regulated intramembrane proteolysis (RIP) to release the N-terminal transcription activating (TA) fragment that enters the nucleus and regulates the expression of target genes. METHODS: To expand our understanding of Creb3l1's role in skeletal development and skeletal patterning, we aimed to generate animals expressing only the TA fragment of Creb3l1 lacking the transmembrane domain and thereby not regulated through RIP. However, the CRISPR/Cas9-mediated genome editing in zebrafish Danio rerio caused a frameshift mutation that added 56 random amino acids at the C-terminus of the TA fragment (TA+), making it unable to enter the nucleus. Thus, TA+ does not regulate transcription, and the creb3l1TA+/TA+ fish do not mediate creb3l1-dependent transcription. RESULTS: We document that the creb3l1TA+/TA+ fish exhibit defects in the patterning of caudal fin lepidotrichia, with significantly distalized points of proximal bifurcation and decreased secondary bifurcations. Moreover, using the caudal fin amputation model, we show that creb3l1TA+/TA+ fish have decreased regeneration and that their regenerates replicate the distalization and bifurcation defects observed in intact fins of creb3l1TA+/TA+ animals. These defects correlate with altered expression of the shha and ptch2 components of the Sonic Hedgehog signaling pathway in creb3l1TA+/TA+ regenerates. CONCLUSION: Together, our results uncover a previously unknown intersection between Creb3l1 and the Sonic Hedgehog pathway and document a novel role of Creb3l1 in tissue patterning.

TGFß signaling is required for sclerotome resegmentation during development of the spinal column in Gallus gallus.

We previously showed the importance of TGFß signaling in development of the mouse axial skeleton. Here, we provide the first direct evidence that TGFß signaling is required for resegmentation of the sclerotome using chick embryos. Lipophilic fluorescent tracers, DiO and DiD, were microinjected into adjacent somites of embryos treated with or without TGFßRI inhibitors, SB431542, SB525334 or SD208, at developmental day E2.5 (HH16). Lineage tracing of labeled cells was observed over the course of 4 days until the completion of resegmentation at E6.5 (HH32). Vertebrae were malformed and intervertebral discs were small and misshapen in inhibitor injected embryos. Hypaxial myofibers were also increased in thickness after treatment with the inhibitor. Inhibition of TGFß signaling resulted in alterations in resegmentation that ranged between full, partial, and slanted shifts in distribution of DiO or DiD labeled cells within vertebrae. Patterning of rostro-caudal markers within sclerotome was disrupted at E3.5 after treatment with TGFßRI inhibitor with rostral domains expressing both rostral and caudal markers. We propose that TGFß signaling regulates rostro-caudal polarity and subsequent resegmentation in sclerotome during spinal column development.

Mechanisms of TGFß in prostaglandin synthesis and sperm guidance in Caenorhabditis elegans.

BACKGROUND: The transparent epidermis of Caenorhabditis elegans makes it an attractive model to study sperm motility and migration within an intact reproductive tract. C elegans synthesize specific F-series prostaglandins (PGFs) that are important for guiding sperm toward the spermatheca. These PGFs are synthesized from polyunsaturated fatty acid (PUFA) precursors, such as arachidonic acid (AA), via a novel pathway, independent of the classical cyclooxygenases (Cox) responsible for most PG synthesis. While the enzyme(s) responsible for PG synthesis has yet to be identified, the DAF-7 TGFß pathway has been implicated in modulating PG levels and sperm guidance. RESULTS: We find that the reduced PGF levels in daf-1 type I receptor mutants are responsible for the sperm guidance defect. The lower level of PGs in daf-1 mutants is due in part to the inaccessibility of AA. Finally, lipid analysis and assessment of sperm guidance in daf-1;daf-3 double mutants suggest DAF-3 suppresses PG production and sperm accumulation at the spermatheca. Our data suggest that DAF-3 functions in the nervous system, and possibly the germline, to affect sperm guidance. CONCLUSION: The C elegans TGFß pathway regulates many pathways to modulate PG metabolism and sperm guidance. These pathways likely function in the nervous system and possibly the germline.

Antagonism of BMP signaling is insufficient to induce fibrous differentiation in primary sclerotome.

Sclerotome is the embryonic progenitor of the axial skeleton. It was previously shown that Tgfbr2 is required in sclerotome for differentiation of fibrous skeletal tissues including the annulus fibrosus of the intervertebral disc. Alternatively, BMP signaling is required to form the vertebral body through chondrogenesis. In addition, TGFß added to sclerotome cultures induces expression of markers for fibrous tissue differentiation but not cartilage or bone. The mechanism of how TGFß signaling regulates this lineage decision in sclerotome is not known and could be due to the production of instructive or inhibitory signals or a combination of the two. Here we show that TGFß antagonizes BMP/ Smad1/5 signaling in primary sclerotome likely through regulation of Noggin, an extracellular BMP antagonist, to prevent chondrogenesis. We then tested whether inhibition of BMP signaling, and inhibition of chondrogenesis, is sufficient to push cells toward the fibrous cell fate. While Noggin inhibited BMP/ Smad1/5 signaling and the formation of chondrogenic nodules in sclerotome cultures; Noggin and inhibition of BMP signaling through Gremlin or DMH2 were insufficient to induce fibrous tissue differentiation. The results suggest inhibition of BMP signaling is not sufficient to stimulate fibrous tissue differentiation and additional signals are likely required. We propose that TGFß has a dual role in regulating sclerotome fate. First, it inhibits BMP signaling potentially through Noggin to prevent chondrogenesis and, second, it provides an unknown instructive signal to promote fibrous tissue differentiation in sclerotome. The results have implications for the design of stem cell-based therapies for skeletal diseases.

Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I.

Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo.

Erg cooperates with TGF-ß to control mesenchymal differentiation.

Transforming growth factor ß (TGF-ß) signaling plays an integral role in skeletal development. Conditional deletion of the TGF-ß type II receptor (Tgfbr2) from type II Collagen (Col2a) expressing cells results in defects in development of the annulus fibrosus (AF) of the intervertebral disc (IVD). We previously used microarray analysis to search for marker genes of AF as well as transcription factors regulated by TGF-ß during AF development. The transcription factor avian erythroblastosis virus E-26 (v-ets) oncogene related (Erg) was identified in the microarray screen as a candidate regulator of AF development. To study the effects of TGF-ß on AF differentiation and the role of Erg in this process, we used mouse sclerotome grown in micromass cultures. At 0.5ng TGF-ß/ml, sclerotome cells started to express markers of AF. Regulation of Erg by TGF-ß was confirmed in these cells. In addition, TGF-ß soaked Affi-gel beads implanted into the axial skeleton of stage HH 25 chick embryos showed that TGF-ß could induce expression of Erg mRNA in vivo. Next, an adenovirus to over-express Erg in primary sclerotome micromass cultures was generated. Over-expression of Erg led to a change in cell morphology and inhibition of differentiation into hyaline cartilage as seen by reduced Alcian blue staining and decreased Sox9 and c-Maf expression. Erg was not sufficient to induce expression of AF markers and expression of Sca1, a marker of pluripotent progenitor cells, was up-regulated in Erg expressing cells. When cells that ectopically expressed Erg were treated with TGF-ß, enhanced expression of specific differentiation markers was observed suggesting Erg can cooperate with TGF-ß to regulate differentiation of the sclerotome. Furthermore, we showed using co-immunopreciptiation that Erg and Smad3 bind to each other suggesting a mechanism for their functional interaction.

Inactivation of Tgfbr2 in Osterix-Cre expressing dental mesenchyme disrupts molar root formation.

It has been difficult to examine the role of TGF-ß in post-natal tooth development due to perinatal lethality in many of the signaling deficient mouse models. To address the role of Tgfbr2 in postnatal tooth development, we generated a mouse in which Tgfbr2 was deleted in odontoblast- and bone-producing mesenchyme. Osx-Cre;Tgfbr2(fl/fl) mice were generated (Tgfbr2(cko)) and post-natal tooth development was compared in Tgfbr2(cko) and control littermates. X-ray and µCT analysis showed that in Tgfbr2(cko) mice radicular dentin matrix density was reduced in the molars. Molar shape was abnormal and molar eruption was delayed in the mutant mice. Most significantly, defects in root formation, including failure of the root to elongate, were observed by postnatal day 10. Immunostaining for Keratin-14 (K14) was used to delineate Hertwig's epithelial root sheath (HERS). The results showed a delay in elongation and disorganization of the HERS in Tgfbr2(cko) mice. In addition, the HERS was maintained and the break up into epithelial rests was attenuated suggesting that Tgfbr2 acts on dental mesenchyme to indirectly regulate the formation and maintenance of the HERS. Altered odontoblast organization and reduced Dspp expression indicated that odontoblast differentiation was disrupted in the mutant mice likely contributing to the defect in root formation. Nevertheless, expression of Nfic, a key mesenchymal regulator of root development, was similar in Tgfbr2(cko) mice and controls. The number of osteoclasts in the bone surrounding the tooth was reduced and osteoblast differentiation was disrupted likely contributing to both root and eruption defects. We conclude that Tgfbr2 in dental mesenchyme and bone is required for tooth development particularly root formation.

PIKE-A is required for prolactin-mediated STAT5a activation in mammary gland development.

PI 3-kinase enhancer A (PIKE-A) is critical for the activation of Akt signalling, and has an essential function in promoting cancer cell survival. However, its physiological functions are poorly understood. Here, we show that PIKE-A directly associates with both signal transducer and activator of transcription 5a (STAT5a) and prolactin (PRL) receptor, which is essential for PRL-provoked STAT5a activation and the subsequent gene transcription. Depletion of PIKE-A in HC11 epithelial cells diminished PRL-induced STAT5 activation and cyclin D1 expression, resulting in profoundly impaired cell proliferation in vitro. To confirm the function of PIKE-A in PRL signalling in vivo, we generated PIKE knockout (PIKE-/-) mice. PIKE-/- mice displayed a severe lactation defect that was characterized by enhanced apoptosis and impaired proliferation of mammary epithelial cells. At parturition, STAT5 activation and cyclin D1 expression were substantially reduced in the mammary epithelium of PIKE-/- mice. The defective mammary gland development in PIKE-/- mice was rescued by overexpression of a mammary-specific cyclin D1 transgene. These data establish a critical function for PIKE-A in mediating PRL functions.

PDGF mediates TGFß-induced migration during development of the spinous process.

Mechanisms mediating closure of the dorsal vertebrae are not clear. Previously, we showed that deletion of TGFß type II receptor (Tgfbr2) in sclerotome in mice results in failure in the formation of the spinous process, mimicking spina bifida occulta, a common malformation in humans. In this study, we aimed to determine whether missing dorsal structures in Tgfbr2 mutant mice were due to defects in mesenchymal migration and to clarify mechanism of TGFß-mediated migration. First, we showed that gross alterations in dorsal vertebrae were apparent by E16.5days in Tgfbr2 mutants. In addition, histological staining showed that the mesenchyme adjacent to the developing cartilage was thin compared to controls likely due to reduced proliferation and migration of these cells. Next, we used a chemotaxis migration assay to show that TGFß promotes migration in mixed cultures of embryonic sclerotome and associated mesenchyme. TGFß stimulated expression of PDGF ligands and receptors in the cultures and intact PDGF signaling was required for TGFß-mediated migration. Since PDGF ligands are expressed in the sclerotome-derived cartilage where Tgfbr2 is deleted and the receptors are predominantly expressed in the adjacent mesenchyme, we propose that TGFß acts on the sclerotome to regulate expression of PDGF ligands, which then act on the associated mesenchyme in a paracrine fashion to mediate proliferation, migration and subsequent differentiation of the adjacent sclerotome.

Disruption of PCP signaling causes limb morphogenesis and skeletal defects and may underlie Robinow syndrome and brachydactyly type B.

Brachydactyly type B (BDB1) and Robinow syndrome (RRS) are two skeletal disorders caused by mutations in ROR2, a co-receptor of Wnt5a. Wnt5a/Ror2 can activate multiple branches of non-canonical Wnt signaling, but it is unclear which branch(es) mediates Wnt5a/Ror2 function in limb skeletal development. Here, we provide evidence implicating the planar cell polarity (PCP) pathway as the downstream component of Wnt5a in the limb. We show that a mutation in the mouse PCP gene Vangl2 causes digit defects resembling the clinical phenotypes in BDB1, including loss of phalanges. Halving the dosage of Wnt5a in Vangl2 mutants enhances the severity and penetrance of the digit defects and causes long bone defects reminiscent of RRS, suggesting that Wnt5a and Vangl2 function in the same pathway and disruption of PCP signaling may underlie both BDB1 and RRS. Consistent with a role for PCP signaling in tissue morphogenesis, mutation of Vangl2 alters the shape and dimensions of early limb buds: the width and thickness are increased, whereas the length is decreased. The digit pre-chondrogenic condensates also become wider, thicker and shorter. Interestingly, altered limb bud dimensions in Vangl2 mutants also affect limb growth by perturbing the signaling network that regulates the balance between Fgf and Bmp signaling. Halving the dosage of Bmp4 partially suppresses the loss of phalanges in Vangl2 mutants, supporting the hypothesis that an aberrant increase in Bmp signaling is the cause of the brachydactyly defect. These findings provide novel insight into the signaling mechanisms of Wnt5a/Ror2 and the pathogenesis in BDB1 and RRS.

TGF-ß2 suppresses macrophage cytokine production and mucosal inflammatory responses in the developing intestine.

BACKGROUND & AIMS: Premature neonates are predisposed to necrotizing enterocolitis (NEC), an idiopathic, inflammatory bowel necrosis. We investigated whether NEC occurs in the preterm intestine due to incomplete noninflammatory differentiation of intestinal macrophages, which increases the risk of a severe mucosal inflammatory response to bacterial products. METHODS: We compared inflammatory properties of human/murine fetal, neonatal, and adult intestinal macrophages. To investigate gut-specific macrophage differentiation, we next treated monocyte-derived macrophages with conditioned media from explanted human fetal and adult intestinal tissues. Transforming growth factor-ß (TGF-ß) expression and bioactivity were measured in fetal/adult intestine and in NEC. Finally, we used wild-type and transgenic mice to investigate the effects of deficient TGF-ß signaling on NEC-like inflammatory mucosal injury. RESULTS: Intestinal macrophages in the human preterm intestine (fetus/premature neonate), but not in full-term neonates and adults, expressed inflammatory cytokines. Macrophage cytokine production was suppressed in the developing intestine by TGF-ß, particularly the TGF-ß(2) isoform. NEC was associated with decreased tissue expression of TGF-ß(2) and decreased TGF-ß bioactivity. In mice, disruption of TGF-ß signaling worsened NEC-like inflammatory mucosal injury, whereas enteral supplementation with recombinant TGF-ß(2) was protective. CONCLUSIONS: Intestinal macrophages progressively acquire a noninflammatory profile during gestational development. TGF-ß, particularly the TGF-ß(2) isoform, suppresses macrophage inflammatory responses in the developing intestine and protects against inflammatory mucosal injury. Enterally administered TGF-ß(2) protected mice from experimental NEC-like injury.

Wnt5a as an effector of TGFß in mammary development and cancer.

Wnt5a is a member of the Wingless-related/MMTV-integration family of secreted growth factors, which are involved in a wide range of cellular processes. Wnt signaling can be broadly divided into two categories the canonical, ß-catenin-dependent pathway and the non-canonical ß-catenin-independent pathway. Wnt5a is a non-canonical signaling member of the Wnt family. Loss of Wnt5a is associated with early relapse of invasive breast cancer, increased metastasis, and poor survival in humans. It has been shown that TGF-ß directly regulates expression of Wnt5a in mammary gland and that Wnt5a mediates the effects of TGF-ß on branching during mammary gland development. Here we review the evidence suggesting Wnt5a acts as an effector of TGF-ß actions in breast cancer. It is suggested that the tumor suppressive functions of TGF-ß involve Wnt5a-mediated antagonism of Wnt/ß-catenin signaling and limiting the stem cell population. Interactions between TGF-ß and Wnt5a in metastasis appear to be more complex, and may depend on specific cues from the microenvironment as well as activation of specific intracellular signaling pathways.

Tgfbr2 in Dental Pulp Cells Guides Neurite Outgrowth in Developing Teeth.

Transforming growth factor ß (TGFß) plays an important role in tooth morphogenesis and mineralization. During postnatal development, the dental pulp (DP) mesenchyme secretes neurotrophic factors that guide trigeminal nerve fibers into and throughout the DP. This process is tightly linked with dentin formation and mineralization. Our laboratory established a mouse model in which Tgfbr2 was conditionally deleted in DP mesenchyme using an Osterix promoter-driven Cre recombinase (Tgfbr2 cko ). These mice survived postnatally with significant defects in bones and teeth, including reduced mineralization and short roots. Hematoxylin and eosin staining revealed reduced axon-like structures in the mutant mice. Reporter imaging demonstrated that Osterix-Cre activity within the tooth was active in the DP and derivatives, but not in neuronal afferents. Immunofluorescence staining for ß3 tubulin (neuronal marker) was performed on serial cryosections from control and mutant molars on postnatal days 7 and 24 (P7, P24). Confocal imaging and pixel quantification demonstrated reduced innervation in Tgfbr2 cko first molars at both stages compared to controls, indicating that signals necessary to promote neurite outgrowth were disrupted by Tgfbr2 deletion. We performed mRNA-Sequence (RNA-Seq) and gene onotology analyses using RNA from the DP of P7 control and mutant mice to investigate the pathways involved in Tgfbr2-mediated tooth development. These analyses identified downregulation of several mineralization-related and neuronal genes in the Tgfbr2 cko DP compared to controls. Select gene expression patterns were confirmed by quantitative real-time PCR and immunofluorescence imaging. Lastly, trigeminal neurons were co-cultured atop Transwell filters overlying primary Tgfbr2 f/f DP cells. Tgfbr2 in the DP was deleted via Adenovirus-expressed Cre recombinase. Confocal imaging of axons through the filter pores showed increased axonal sprouting from neurons cultured with Tgfbr2-positive DP cells compared to neurons cultured alone. Axon sprouting was reduced when Tgfbr2 was knocked down in the DP cells. Immunofluorescence of dentin sialophosphoprotein in co-cultured DP cells confirmed reduced mineralization potential in cells with Tgfbr2 deletion. Both our proteomics and RNA-Seq analyses indicate that axonal guidance cues, particularly semaphorin signaling, were disrupted by Tgfbr2 deletion. Thus, Tgfbr2 in the DP mesenchyme appears to regulate differentiation and the cells' ability to guide neurite outgrowth during tooth mineralization and innervation.

Misexpression of wingless-related MMTV integration site 5A in mouse mammary gland inhibits the milk ejection response and regulates connexin43 phosphorylation.

Wingless-related MMTV integration site 5A (Wnt5a) is a noncanonical signaling WNT that is expressed in every stage of mouse mammary gland development except lactation. Using slow release pellets containing WNT5A as well as Wnt5a-null tissue, we previously showed that WNT5A acts to limit mammary development. Here, we generated transgenic mice that overexpress WNT5A in the mammary epithelium using the mouse mammary tumor virus promoter (M5a mice). Lactation was impaired in two high WNT5A-expressing lines. Lactation defects could not be explained by differences in apoptosis, lineage differentiation, milk synthesis, or secretion. Instead, misexpression of WNT5A led to a failure in oxytocin response and milk ejection. Noting the similarity between the M5a phenotype and that of mice with a mutation in connexin43 (Cx43; official gene symbol Gja1), we examined Cx43 phosphorylation and localization in M5a mice. In wild-type mice, Cx43 switched from a phosphorylated to a more hypophosphorylated form after parturition. In contrast, the phosphorylated form of Cx43 was maintained after parturition in M5a mice. Using a nontumorigenic breast cell line, MCF10A, we showed that, in addition to increasing the levels of phosphorylation of Cx43 on serine-368, ectopic expression of WNT5A reduced or blocked the amount of dye transferred between cells. In summary, we propose that WNT5A inhibits the response to oxytocin and prevents milk ejection through regulation of Cx43 function.

Chondrocyte-specific regulatory activity of Runx2 is essential for survival and skeletal development.

Coordinated activities of multiple mesenchymal cell types contribute to the development of the mammalian skeleton formed through endochondral ossification. Synthesis of a cartilage template by chondrocytes is an obligatory step for the generation of skeletal elements during endochondral ossification. Gene ablation studies have established that Runx2 is an essential transcription factor for bone formation and the differentiation of skeletal cells. However, global gene deletion has failed to discern the tissue- and cell type-specific roles of Runx2. We generated floxed mice to elucidate the Runx2 regulatory control distinctive to cartilage tissue during bone development. Exon 8 of the Runx2 gene was selectively deleted in developing chondrocytes by utilizing Col2a-Cre mice. Cell- and tissue-specific gene recombination was confirmed by ß-gal activity in R26R mice. The chondrocyte-specific loss of Runx2 caused failure of endochondral ossification, impaired craniofacial development, dwarfism, and perinatal lethality. Radiographic imaging and histochemical approaches were used to characterize the skeletal phenotype. We conclude that regulatory control of Runx2 in chondrocytes is essential for endochondral ossification, and it is independent of the role of Runx2 in osteoblasts.

TGF-beta promotes Th17 cell development through inhibition of SOCS3.

TGF-beta, together with IL-6 and IL-21, promotes Th17 cell development. IL-6 and IL-21 induce activation of STAT3, which is crucial for Th17 cell differentiation, as well as the expression of suppressor of cytokine signaling (SOCS)3, a major negative feedback regulator of STAT3-activating cytokines that negatively regulates Th17 cells. However, it is still largely unclear how TGF-beta regulates Th17 cell development and which TGF-beta signaling pathway is involved in Th17 cell development. In this report, we demonstrate that TGF-beta inhibits IL-6- and IL-21-induced SOCS3 expression, thus enhancing as well as prolonging STAT3 activation in naive CD4(+)CD25(-) T cells. TGF-beta inhibits IL-6-induced SOCS3 promoter activity in T cells. Also, SOCS3 small interfering RNA knockdown partially compensates for the action of TGF-beta on Th17 cell development. In mice with a dominant-negative form of TGF-beta receptor II and impaired TGF-beta signaling, IL-6-induced CD4(+) T cell expression of SOCS3 is higher whereas STAT3 activation is lower compared with wild-type B6 CD4(+) T cells. The addition of a TGF-beta receptor I kinase inhibitor that blocks Smad-dependent TGF-beta signaling greatly, but not completely, abrogates the effect of TGF-beta on Th17 cell differentiation. Our data indicate that inhibition of SOCS3 and, thus, enhancement of STAT3 activation is at least one of the mechanisms of TGF-beta promotion of Th17 cell development.

Tgfbr2 is required for development of the skull vault.

Transforming growth factor beta (TGFbeta) is known to play important roles in multiple developmental processes. One of the main functions is in skeletal development. Our previous studies demonstrated that loss of Tgfbr2 in Prx1Cre-expressing limb mesenchyme results in defects in the long bones and joints of mice. Here we show that loss of Tgfbr2 also results in defects in the development of the skull vault indicating Tgfbr2 has a critical role in intramembranous bone formation as well as endochondral bone formation. Mutant mice did not survive after birth and demonstrated an open skull. The first signs of skull defects were observed at E14.5 day. Prx1Cre(+)/Tgfbr2(f/f) embryos showed significantly reduced cell proliferation in the developing mesenchyme of the skull by E14.5 day without any detectable alteration in apoptosis suggesting that reduced cell proliferation in Prx1Cre(+)/Tgfbr2(f/f) embryos was at least partially responsible for the defects observed. Immunofluorescent staining showed a significant reduction in the expression of Runx2/Cbfa1 and Osterix/Sp7 in Prx1Cre(+)/Tgfbr2(f/f) embryos suggesting that osteoblast differentiation was also altered in Prx1Cre(+)/Tgfbr2(f/f) embryos. To distinguish between the effects of losing Tgfbr2 on mesenchymal proliferation versus osteoblast differentiation, osteoprogenitor cells from the skulls of Tgfbr2(f/f) embryos were cultured under conditions of high cell density and Tgfbr2 was deleted from the cells using Adeno-Cre virus. RT-PCR analysis showed that the mRNA level of Runx2 and Osterix as well as Dlx5 and Msx2 were down-regulated in Tgfbr2-deleted cultures compared to control cultures indicating that Tgfbr2 regulates osteoblast differentiation independent of regulating proliferation. Together, these results suggest that Tgfbr2 is required for normal development of the skull.

Molecular profiling of the developing mouse axial skeleton: a role for Tgfbr2 in the development of the intervertebral disc.

BACKGROUND: Very little is known about how intervertebral disc (IVD) is formed or maintained. Members of the TGF-beta superfamily are secreted signaling proteins that regulate many aspects of development including cellular differentiation. We recently showed that deletion of Tgfbr2 in Col2a expressing mouse tissue results in alterations in development of IVD annulus fibrosus. The results suggested TGF-beta has an important role in regulating development of the axial skeleton, however, the mechanistic basis of TGF-beta action in these specialized joints is not known. One of the hurdles to understanding development of IVD is a lack of known markers. To identify genes that are enriched in the developing mouse IVD and to begin to understand the mechanism of TGF-beta action in IVD development, we undertook a global analysis of gene expression comparing gene expression profiles in developing mouse vertebrae and IVD. We also compared expression profiles in tissues from wild type and Tgfbr2 mutant mice as well as in sclerotome cultures treated with TGF-beta or BMP4. RESULTS: Lists of IVD and vertebrae enriched genes were generated. Expression patterns for several genes were verified either through in situ hybridization or literature/database searches resulting in a list of genes that can be used as markers of IVD. Cluster analysis using genes listed under the Gene Ontology terms multicellular organism development and pattern specification indicated that mutant IVD more closely resembled vertebrae than wild type IVD. We also generated lists of genes regulated by TGF-beta or BMP4 in cultured sclerotome. As expected, treatment with BMP4 resulted in up-regulation of cartilage marker genes including Acan, Sox 5, Sox6, and Sox9. In contrast, treatment with TGF-beta1 did not regulate expression of cartilage markers but instead resulted in up-regulation of many IVD markers including Fmod and Adamtsl2. CONCLUSIONS: We propose TGF-beta has two functions in IVD development: 1) to prevent chondrocyte differentiation in the presumptive IVD and 2) to promote differentiation of annulus fibrosus from sclerotome. We have identified genes that are enriched in the IVD and regulated by TGF-beta that warrant further investigation as regulators of IVD development.

Scaffoldless tissue-engineered cartilage for studying transforming growth factor beta-mediated cartilage formation.

Reduced transforming growth factor beta (TGF-ß) signaling is associated with osteoarthritis (OA). TGF-ß is thought to act as a chondroprotective agent and provide anabolic cues to cartilage, thus acting as an OA suppressor in young, healthy cartilage. A potential approach for treating OA is to identify the factors that act downstream of TGF-ß's anabolic pathway and target those factors to promote cartilage regeneration or repair. The aims of the present study were to (a) develop a scaffoldless tissue-engineered cartilage model with reduced TGF-ß signaling and disrupted cartilage formation and (b) validate the system for identifying the downstream effectors of TGF-ß that promote cartilage formation. Sox9 was used to validate the model because Sox9 is known to promote cartilage formation and TGF-ß regulates Sox9 activity. Primary bovine articular chondrocytes were grown in Transwell supports to form cartilage tissues. An Alk5/TGF-ß type I receptor inhibitor, SB431542, was used to attenuate TGF-ß signaling, and an adenovirus encoding FLAG-Sox9 was used to drive the expression of Sox9 in the in vitro-generated cartilage. SB431542-treated tissues exhibited reduced cartilage formation including reduced thicknesses and reduced proteoglycan staining compared with control tissue. Expression of FLAG-Sox9 in SB431542-treated cartilage allowed the formation of cartilage despite antagonism of the TGF-ß receptor. In summary, we developed a three-dimensional in vitro cartilage model with attenuated TGF-ß signaling. Sox9 was used to validate the model for identification of anabolic agents that counteract loss of TGF-ß signaling. This model has the potential to identify additional anabolic factors that could be used to repair or regenerate damaged cartilage.