Controlled release of bioactive IL-2 from visible light photocured biodegradable elastomers for cancer immunotherapy applications.

Biodegradable elastomeric controlled-release poly (decane-co-tricarballylate) (PDET) based matrices capable of maintaining the stability and bioactivity of Interleukin-2 (IL-2) through the utilization of visible-light curing and solvent-free loading of the cytokine are reported. The elastomeric devices were fabricated by intimately mixing lyophilized IL-2 powder with the acrylated prepolymer before photocrosslinking. The bioactivity of the released protein was assessed by its ability to stimulate the proliferation of the C57BL/6 mouse cytotoxic T lymphocyte, and its concentration was analysed using ELISA. The influence of changes in the polymer's physicochemical and mechanical properties on IL-2 release kinetics and bioactivity were also studied. The increase in the device's surface area and the incorporation of trehalose in the loaded lyophilized mix increased the IL-2 release rate with drug release proceeding via typical zero-order release kinetics. Moreover, the decrease in the degree of acrylation of the prepared devices increased the IL-2 release rate. The bioactivity assay showed that IL-2 retained over 94% of its initial bioactivity throughout 28 days of the release period. A new protein delivery vehicle composed of biodegradable PDET elastomers was demonstrated to be promising and effective for linear, constant, and sustained osmotic-driven release of bioactive IL-2 and other sensitive proteins and hormones.

Learning-induced mRNA alterations in olfactory bulb mitral cells in neonatal rats.

In the olfactory bulb, a cAMP/PKA/CREB-dependent form of learning occurs in the first week of life that provides a unique mammalian model for defining the epigenetic role of this evolutionarily ancient plasticity cascade. Odor preference learning in the week-old rat pup is rapidly induced by a 10-min pairing of odor and stroking. Memory is demonstrable at 24 h, but not 48 h, posttraining. Using this paradigm, pups that showed peppermint preference 30 min posttraining were sacrificed 20 min later for laser microdissection of odor-encoding mitral cells. Controls were given odor only. Microarray analysis revealed that 13 nonprotein-coding mRNAs linked to mRNA translation and splicing and 11 protein-coding mRNAs linked to transcription differed with odor preference training. MicroRNA23b, a translation inhibitor of multiple plasticity-related mRNAs, was down-regulated. Protein-coding transcription was up-regulated for Sec23b, Clic2, Rpp14, Dcbld1, Magee2, Mstn, Fam229b, RGD1566265, and Mgst2. Gng12 and Srcg1 mRNAs were down-regulated. Increases in Sec23b, Clic2, and Dcbld1 proteins were confirmed in mitral cells in situ at the same time point following training. The protein-coding changes are consistent with extracellular matrix remodeling and ryanodine receptor involvement in odor preference learning. A role for CREB and AP1 as triggers of memory-related mRNA regulation is supported. The small number of gene changes identified in the mitral cell input/output link for 24 h memory will facilitate investigation of the nature, and reversibility, of changes supporting temporally restricted long-term memory.

Histone deacetylase inhibition induces odor preference memory extension and maintains enhanced AMPA receptor expression in the rat pup model.

Histone deacetylase (HDAC) plays a role in synaptic plasticity and long-term memory formation. We hypothesized that trichostatin-A (TSA), an HDAC inhibitor, would promote long-term odor preference memory and maintain enhanced GluA1 receptor levels that have been hypothesized to support memory. We used an early odor preference learning model in neonate rat pups that normally produces only 24-h memory to test behavior and examine receptor protein expression. Our behavioral studies showed that intrabulbar infusion of TSA, prior to pairing of the conditioned stimulus (peppermint odor) with the unconditioned stimulus (tactile stimulation), prolonged 24-h odor preference memory for at least 9 d. The prolonged odor preference memory was selective for the paired odor and was also observed using a specific HDAC6 inhibitor, tubacin, supporting a role for histone acetylation in associative memory. Immunoblot analysis showed that GluA1 receptor membrane expression in the olfactory bulbs of the TSA-treated group was significantly increased at 48 h unlike control rats without TSA. Immunohistochemistry revealed significant increase of GluA1 expression in olfactory bulb glomeruli 5 d after training. These results extend previous evidence for a close relationship between enhanced GluA1 receptor membrane expression and memory expression. Together, these findings provide a new single-trial appetitive model for understanding the support and maintenance of memories of varying duration.

Cav1.2, but not Cav1.3, L-type calcium channel subtype mediates nicotine-induced conditioned place preference in mice.

Nicotine use is one of the most common forms of drug addiction. Although L-type calcium channels (LTCCs) are involved in nicotine addiction, the contribution of the two primary LTCC subtypes (Cav1.2 and 1.3) is unknown. This study aims to determine the contribution of these two LTCC subtypes to nicotine-induced conditioned place preference (CPP) responses by using transgenic mouse models that do not express Cav1.3 (Cav1.3-/-) or contain a mutation in the dihydropyridine (DHP) site of the Cav1.2 (Cav1.2DHP-/-). We found a hyperbolic dose dependent nicotine (0.1-1mg/kg; 0.5mg/kg optimum) effect on place preference in wild type (WT) mice, that could be prevented by the DHP LTCC blocker nifedipine pretreatment. Similarly, Cav1.3-/- mice showed nicotine-induced place preference which was antagonized by nifedipine. In contrast, nifedipine pretreatment of Cav1.2DHP-/- mice had no effect on nicotine-induced CPP responses, suggesting an involvement of Cav1.2 subtype in the nicotine-induced CPP response. Nifedipine alone failed to produce either conditioned place aversion or CPP in WT mice. These results collectively indicate Cav1.2, but not Cav1.3 LTCC subtype regulates, at least in part, the reinforcing effects of nicotine use.

Overexpression of MMP13 in human osteoarthritic cartilage is associated with the SMAD-independent TGF-ß signalling pathway.

INTRODUCTION: In vitro and animal model of osteoarthritis (OA) studies suggest that TGF-ß signalling is involved in OA, but human data is limited. We undertook this study to elucidate the role of TGF-ß signalling pathway in OA by comparing the expression levels of TGFB1 and BMP2 as ligands, SMAD3 as an intracellular mediator, and MMP13 as a targeted gene between human osteoarthritic and healthy cartilage. METHODS: Human cartilage samples were collected from patients undergoing total hip/knee joint replacement surgery due to primary OA or hip fractures as controls. RNA was extracted from the cartilage tissues. Real-time quantitative PCR was performed to measure gene expression. Mann-Whitney test was utilized to compare the expression levels of TGFB1, BMP2, SMAD3 and MMP13 in human cartilage between OA cases and controls. Spearman's rank correlation coefficient (rho) was calculated to examine the correlation between the expression levels of the four genes studied and non-parametric regression was used to adjust for covariates. RESULTS: A total of 32 OA cases (25 hip OA and 7 knee OA) and 21 healthy controls were included. The expression of TGFB1, SMAD3, and MMP13 were on average 70%, 46%, and 355% higher, respectively, whereas the expression of BMP2 was 88% lower, in OA-affected cartilage than that of controls (all p < 0.03), but no difference was observed between hip and knee OA (all p > 0.4). The expression of TGFB1 was correlated with the expression of SMAD3 (rho = 0.50, p = 0.003) and MMP13 (rho = 0.46, p = 0.007) in OA-affected cartilage and the significance became stronger after adjustment for age, sex, and BMI. The expression of BMP2 was negatively correlated with both TGFB1 (rho = -0.50, p = 0.02) and MMP13 (rho = -0.48, p = 0.02) in healthy cartilage, but the significance was altered after adjustment for the covariates. There was no correlation between the expression of SMAD3 and MMP13. CONCLUSIONS: Our results demonstrate that MMP13 expression is associated with an increased expression of TGFB1 in OA-affected cartilage, possibly through SMAD-independent TGF-ß pathway. Furthermore, TGF-ß/SMAD3 is overactivated in OA cartilage; yet, the consequence of this overactivation remains to be established.

TGF-ß signal transduction pathways and osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis, resulting in substantial disability and economic burden worldwide. While its exact pathogenesis remains elusive, both in vitro and human population-based studies have merged to support the hypothesis that TGF-ß/BMP-mediated signalling pathways play a role in the development of OA. Unraveling the TGF-ß/BMP-mediated mechanism(s) in OA has great potential in identifying novel targets and developing new drugs for OA treatment. This review summarizes both in vitro and in vivo evidence of TGF-ß/BMP-mediated signal transduction pathways in OA and discusses the future research direction in this regard.

Cav1.2 and Cav1.3 L-type calcium channels regulate dopaminergic firing activity in the mouse ventral tegmental area.

Dopaminergic projections from the ventral tegmental area (VTA) constitute the mesolimbocortical system that underlies addiction and psychosis primarily as a result of increased dopaminergic transmission. Dopamine release is spike dependent. L-type calcium channels (LTCCs) play an important role in regulating firing activities, but the contribution of specific subtypes remains unclear. This article describes different functions of Cav1.2 and Cav1.3 subtypes in regulating firing properties with two transgenic mouse strains. For basal firing, Cav1.3-deficient (Cav1.3(-/-)) mice had a lower basal firing frequency. The dihydropyridine (DHP) channel blocker nifedipine reduced single-spike firing in mice expressing DHP-insensitive Cav1.2 channels (Cav1.2DHP(-/-) mice), confirming the significant contribution from the Cav1.3 subtype in basal firing. Moreover, the DHP channel activator (S)-(-)-Bay K8644 and the non-DHP channel activator FPL 64176 converted firing patterns from single spiking to bursting in Cav1.2DHP(-/-) mice. Nifedipine inhibited burst firing induced by both activators, suggesting that Cav1.3 also serves an essential role in burst firing. However, FPL 64176 also induced bursting in Cav1.3(-/-) mice. These results indicate that the Cav1.3 subtype is crucial to regulation of basal single-spike firing, while activation of both Cav1.2 and Cav1.3 can support burst firing of VTA neurons.

Tgf-beta induced Erk phosphorylation of smad linker region regulates smad signaling.

The Transforming Growth Factor-Beta (TGF-ß) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-ß binding to a Serine/Threonine kinase receptor complex causes the recruitment and subsequent activation of transcription factors known as smad2 and smad3. These proteins subsequently translocate into the nucleus to negatively or positively regulate gene expression. In this study, we define a second signaling pathway leading to TGF-ß receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-ß induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-ß stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-ß induced fibroblast replication. In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription. Together, these data show that in mesenchymal cell types the TGF-ß/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-ß-induced growth and is part of an integrated signaling web containing multiple interacting pathways rather than discrete smad/non-smad pathways.

Synthesis, characterization and cytocompatibility of a poly(diol-tricarballylate) visible light photo-cross-linked biodegradable elastomer.

The synthesis, characterization and in vitro cytocompatibility of a new family of photo-cross-linked amorphous poly(diol-tricarballylate) (PDT) biodegradable elastomeric polyesters are reported. The synthesis was based on the polycondensation reaction between tricarballylic acid and alkylene diols, followed by acrylation. The prepared and acrylated poly(diol-tricarballylate) (APDT) was characterized by means of FT-IR, (1)H-NMR, GPC and DSC. Liquid-to-solid photo-curing was carried out by exposing the APDT to visible light in the presence of camphorquinone as a photoinitiator. The thermal properties, mechanical characteristics, sol content, long-term in vitro degradation and cytocompatibility of the prepared PDT elastomers were also reported. The mechanical and degradation properties of this new photocurable elastomer can be precisely controlled by varying the density of acrylate moieties in the matrix of the polymer, and through changes in the pre-polymer chain length. The use of visible light cross-linking, possibility of solventless drug loading, controllable mechanical properties and cytocompatibility of these new elastomers make them excellent candidates for use in controlled implantable drug-delivery systems of protein drugs and other biomedical applications.

Dysregulation of kallikrein-related peptidases in renal cell carcinoma: potential targets of miRNAs.

Renal cell carcinoma (RCC) accounts for 3% of all adult malignancies and currently no diagnostic marker exists. Kallikrein-related peptidases (KLKs) have been implicated in numerous cancers including ovarian, prostate, and breast carcinoma. KLKs 5, 6, 10, and 11 have decreased expression in RCC when compared to normal kidney tissue. Our bioinformatic analysis indicated that the KLK 1, 6, and 7 genes have decreased expression in RCC. We experimentally verified these results and found that decreased expression of KLKs 1 and 3 were significantly associated with the clear cell RCC subtype (p<0.001). An analysis of miRNAs differentially expressed in RCC showed that 61 of the 117 miRNAs that were reported to be dysregulated in RCC were predicted to target KLKs. We experimentally validated two targets using two independent approaches. Transfection of miR-224 into HEK-293 cells resulted in decreased KLK1 protein levels. A luciferase assay demonstrated that hsa-let-7f can target KLK10 in the RCC cell line ACHN. Our results, showing differential expression of KLKs in RCC, suggest that KLKs could be novel diagnostic markers for RCC and that their dysregulation could be under miRNA control. The observation that KLKs could represent targets for miRNAs suggests a post-transcriptional regulatory mechanism with possible future therapeutic applications.

Troubleshooting tissue specificity and antibody selection: Procedures in immunohistochemical studies.

Optimal antigen detection and identification is dependent on the tissue of interest, the method of fixation, processing, and antibody specificity. We evaluated specific antigens in frozen middle cerebral artery (MCA) sections from rat brains under various conditions of fixation and differing primary and secondary antibody concentrations. Fresh MCAs were frozen, cryosectioned (8 microm), and adhered to chrom-alum coated slides. The effects of different fixation and antigen retrieval/pretreatments were tested for detection of enzymes and receptors involved in MCA tone regulation. Antigen localization was determined with specific primary antibodies and detected using fluorochrome-conjugated secondary antibodies. Spatial distribution of localized antigens was imaged using confocal microscopy. Frozen sections preserved the morphology of the endothelium and/or vessel wall within the tissue in a manner comparable to formalin-fixed sections. Fixation and tissue processing methods were modified based on the primary antibody used. Optimal antigen detection was obtained using fixatives such as 4% paraformaldehyde, 100% acetone or 100% methanol. Pretreatments, such as 1% SDS, enzymatic digestion using 0.1% trypsin, or application of heat were used to optimize antigen-antibody interaction. Stringent background and control checks were performed to ensure specificity of staining in both single and multiple labeling techniques. In a research setting where epitope detection is not used for diagnostic purposes, there is more latitude in tissue fixation. Frozen samples offer a more versatile method of linking the appropriate fixation and tissue processing to the primary antibody's unique needs. At the same time, it stabilizes the tissue in a format that allows for later analysis of multiple antigens with specific detection requirements in same tissue.

Human kallikrein related peptidases 6 and 13 in combination withCA125 is a more sensitive test for ovarian cancer than CA125 alone.

The current biomarker for ovarian cancer, CA125, lacks the sensitivity and specificity required to detect early stage ovarian cancers. Since several Kallikreins (KLKs) are up regulated in ovarian cancer, they represent a potential pool of biomarkers for ovarian cancer. The purpose of this study is to determine if elevated expression levels of Muc16 (CA125 gene), KLK6 and KLK13 represent a more sensitive test for detection of early stage ovarian cancer than Muc16 alone. Using quantitative real-time PCR, 106 sporadic ovarian tumors and 8 normal ovaries were evaluated for mRNA expression. Analysis for increased expression levels, above controls, of either KLK6, KLK13 or Muc16 improved overall sensitivity to 93%, from 82% for Muc16 alone. Likewise, the negative predictive value increased from 27% to 50% (Muc16 alone compared to combined). With early stage cancers (n=32), both sensitivity increased 50-56% (individually) to 72% (combined), and negative predictive value increased (30% Muc16 to 58% combined). These results show a combined panel of KLK6, KLK13, and Muc16, is a more sensitive test to detect early stage ovarian cancer than Muc16 alone, indicating assaying for several kallikrein-related peptidases, in addition to CA125, could provide a significant advantage to detect ovarian cancer in the early stages.

Brain-derived neurotrophic factor contributes to recovery of skilled reaching after focal ischemia in rats.

BACKGROUND AND PURPOSE: Brain-derived neurotrophic factor (BDNF) is involved in neuronal survival, synaptic plasticity, learning and memory, and neuroplasticity. Further, exogenous treatment with BDNF or exposing animals to enrichment and exercise regimens, which also increase BDNF, enhances behavioral recovery after brain injury. Thus, the beneficial effects of rehabilitation in promoting recovery after stroke may also depend on BDNF. We tested this hypothesis by evaluating the contribution of BDNF to motor skill relearning after endothelin-1-induced middle cerebral artery occlusion in rats. METHODS: Antisense BDNF oligonucleotide, which blocks the expression of BDNF (or saline vehicle) was infused into the contralateral lateral ventricle for 28 days after ischemia. Animals received either a graduated rehabilitation program, including running exercise and skilled reaching training, which simulates clinical practice, or no rehabilitation. Functional recovery was assessed with a battery of tests that measured skilled reaching, forelimb use asymmetry, and foraging ability. RESULTS: Rehabilitation significantly improved skilled reaching ability in the staircase task. Antisense BDNF oligonucleotide effectively blocked BDNF mRNA, and negated the beneficial effects of rehabilitation on recovery of skilled reaching. Importantly, antisense BDNF oligonucleotide did not affect reaching with the unaffected limb, body weight, infarct size, or foraging ability, indicating the treatment was specific to relearning of motor skill after ischemia. CONCLUSIONS: This study is the first to identify a critical role for BDNF in rehabilitation-induced recovery after stroke, and our results suggest that new treatments to enhance BDNF would constitute a promising therapy for promoting recovery of function after stroke.

Barrett's esophagus and cardiac intestinal metaplasia: two conditions within the same spectrum.

BACKGROUND: Immunostaining for cytokeratin 7 (CK7) and cytokeratin 20 (CK20) has a characteristic pattern in Barrett's esophagus (BE), but reports regarding its sensitivity and specificity are inconsistent. Intestinal metaplasia of the gastric cardia (CIM) is histologically similar to BE, but with no abnormal endoscopic findings. OBJECTIVES: To evaluate the sensitivity and specificity of a semi-quantitative CK7/CK20 immunostaining pattern for the diagnosis of BE, and to further elucidate the pathogenesis of CIM. METHODS: Tissues were examined by hematoxylin and eosin and periodic acid schiff/alcian blue stains, and then were immunostained with CK7 and CK20 antibodies. Correlations with other clinical parameters were statistically analyzed. RESULTS: When values were revised based on follow-up data and auxiliary testing, all BE cases (100%) displayed the characteristic BE CK7/CK20 immunostaining pattern, compared with 66% of CIM cases. In the subgroup of patients who were endoscopically and immunohistochemistry-positive but histologically negative, all patients except for one had documented BE when clinical history, auxiliary testing and follow-up were evaluated. There were no statistically significant differences between BE and CIM regarding Helicobacter pylori infection or the type of metaplasia (complete versus incomplete). The sensitivity of the CK7/CK20 pattern reached 100% in the subgroup of CIM patients with a history of acid reflux. Of 26 cases of CIM where follow-up was available, four cases (15%) progressed to BE, and one developed dysplasia. All four cases showed the BE pattern of CK7/CK20 staining and were negative for H pylori infection. CONCLUSIONS: A semiquantitative CK7/CK20 pattern can be used to confirm BE even in the absence of histological evidence. The subgroup of CIM with acid reflux may develop into BE and may need closer follow-up.

Endurance exercise facilitates relearning of forelimb motor skill after focal ischemia.

Endurance exercise (i.e. running), by up-regulating brain-derived neurotrophic factor (BDNF) and other modulators of synaptic plasticity, improves attention and learning, both critical components of stroke rehabilitation. We hypothesized that, following middle cerebral artery occlusion in male Sprague-Dawley rats, endurance exercise would act synergistically with a challenging skilled forelimb task to facilitate motor recovery. Animals were randomly assigned to one of four rehabilitation conditions: no rehabilitation, running only, reach training only, and reach training preceded by running (run/reach training) for 5 weeks beginning 5 days after stroke. The behavioral outcome, morphological change and mRNA expression of proteins implicated in neuroplasticity (BDNF, synapsin I and microtubule-associated protein 2) were compared. Endurance exercise on a motorized running wheel, prior to reach training, enhanced recovery of skilled reaching ability but did not transfer to gross motor skills such as postural support (forelimb asymmetry test) and gait (ladder rung walking test). Microtubule-associated protein 2 staining density in the run/reach group was slightly enhanced in the contralateral motor cortex compared with the contralateral sensory and ipsilateral cingulate cortices, suggesting that running preceding reach training may have resulted in more dendritic branching within the motor cortex in this group. No significant differences in mRNA levels were detected among the training paradigms; however, there was a trend toward greater BDNF and synapsin I mRNA in the reaching groups. These findings suggest that exercise facilitates learning of subsequent challenging reaching tasks after stroke, which has the potential to optimize outcomes in patients with stroke.

Calcium influx through L-type channels generates protein kinase M to induce burst firing of dopamine cells in the rat ventral tegmental area.

Enhanced activity of the dopaminergic system originating in the ventral tegmental area is implicated in addictive and psychiatric disorders. Burst firing increases dopamine levels at the synapse to signal novelty and salience. We have previously reported a calcium-dependent burst firing of dopamine cells mediated by L-type channels following cholinergic stimulation; this paper describes a cellular mechanism resulting in burst firing following L-type channel activation. Calcium influx through L-type channels following FPL 64176 or (S)-(-)-Bay K8644 induced burst firing independent of dopamine, glutamate, or calcium from the internal stores. Burst firing induced as such was completely blocked by the substrate site protein kinase C (PKC) inhibitor chelerythrine but not by the diacylglycerol site inhibitor calphostin C. Western blotting analysis showed that FPL 64176 and (S)-(-)-Bay K8644 increased the cleavage of PKC to generate protein kinase M (PKM) and the specific calpain inhibitor MDL28170 blocked this increase. Prevention of PKM production by inhibiting calpain or depleting PKC blocked burst firing induction whereas direct loading of purified PKM into cells induced burst firing. Activation of the N-methyl-D-aspartic acid type glutamate or cholinergic receptors known to induce burst firing increased PKM expression. These results indicate that calcium influx through L-type channels activates a calcium-dependent protease that cleaves PKC to generate constitutively active and labile PKM resulting in burst firing of dopamine cells, a pathway that is involved in glutamatergic or cholinergic modulation of the central dopamine system.

Transforming growth factor-beta activation of phosphatidylinositol 3-kinase is independent of Smad2 and Smad3 and regulates fibroblast responses via p21-activated kinase-2.

Transforming growth factor-beta (TGF-beta) stimulates cellular proliferation and transformation to a myofibroblast phenotype in vivo and in a subset of fibroblast cell lines. As the Smad pathway is activated by TGF-beta in essentially all cell types, it is unlikely to be the sole mediator of cell type-specific outcomes to TGF-beta stimulation. In the current study, we determined that TGF-beta receptor signaling activates phosphatidylinositol 3-kinase (PI3K) in several fibroblast but not epithelial cultures independently of Smad2 and Smad3. PI3K activation occurs in the presence of dominant-negative dynamin and is required for p21-activated kinase-2 kinase activity and the increased proliferation and morphologic change induced by TGF-beta in vitro.

Internalization-dependent and -independent requirements for transforming growth factor beta receptor signaling via the Smad pathway.

Members of the transforming growth factor beta (TGF-beta) family of proteins signal through cell surface transmembrane serine/threonine protein kinases known as type I and type II receptors. The TGF-beta signal is extended through phosphorylation of receptor-associated Smad proteins by the type I receptor. Although numerous investigations have established the sequence of events in TGF-beta receptor (TGF-beta R) activation, none have examined the role of the endocytic pathway in initiation and/or maintenance of the signaling response. In this study we investigated whether TGF-beta R internalization modulates type I receptor activation, the formation of a functional receptor/Smad/SARA complex, Smad2/3 phosphorylation or nuclear translocation, and TGF-beta-dependent reporter gene activity. Our data provide evidence that, whereas type I receptor phosphorylation and association of SARA and Smad2 with the TGF-beta R complex take place independently of clathrin lattice formation, Smad2 or Smad3 activation and downstream signaling only occur after endocytic vesicle formation. Thus, TGF-beta R endocytosis is not simply a way to dampen the signaling response but instead is required to propagate signaling via the Smad pathway.