Vimentin binding to phosphorylated Erk sterically hinders enzymatic dephosphorylation of the kinase

Cleavage fragments of de novo synthesized vimentin were recently reported to interact with phosphorylated Erk1 and Erk2 MAP kinases (pErk) in injured sciatic nerve, thus linking pErk to a signaling complex retrogradely transported on importins and dynein. Here we clarify the structural basis for this interaction, which explains how pErk is protected from dephosphorylation while bound to vimentin. Pull-down and ELISA experiments revealed robust calcium-dependent binding of pErk to the second coiled-coil domain of vimentin, with observed affinities of binding increasing from 180 nM at 0.1 microM calcium to 15 nM at 10 microM calcium. In contrast there was little or no binding of non-phosphorylated Erk to vimentin under these conditions. Geometric and electrostatic complementarity docking generated a number of solutions wherein vimentin binding to pErk occludes the lip containing the phosphorylated residues in the kinase. Binding competition experiments with Erk peptides confirmed a solution in which vimentin covers the phosphorylation lip in pErk, interacting with residues above and below the lip. The same peptides inhibited pErk binding to the dynein complex in sciatic nerve axoplasm, and interfered with protection from phosphatases by vimentin. Thus, a soluble intermediate filament fragment interacts with a signaling kinase and protects it from dephosphorylation by calcium-dependent steric hindrance.     

Intermediate filament proteins participate in signal transduction

How timely transport of chemical signals between the distal end of long axonal processes and the cell bodies of neurons occurs is an interesting and unresolved issue. Recently, Perlson et al. presented evidence that cleavage products of newly synthesized vimentin, an intermediate filament (IF) protein, interact with mitogen-activated protein (MAP) kinases at sites of axon injury. These IF fragments appear to be required for the transport of these kinases to the cell body along microtubule tracks. The truncated vimentin is instrumental in signal propagation as it provides a scaffold that brings together activated MAP kinases (such as Erk 1 and Erk2), as well as importin beta and cytoplasmic dynein. The authors propose that this all-in-one transport complex has the extraordinary ability to travel towards the cell body and enter the nucleus where the kinases activate and influence gene expression so that a neuron can generate a timely response to injury.     

Localized regulation of axonal RanGTPase controls retrograde injury signaling in peripheral nerve

Peripheral sensory neurons respond to axon injury by activating an importin-dependent retrograde signaling mechanism. How is this mechanism regulated? Here, we show that Ran GTPase and its associated effectors RanBP1 and RanGAP regulate the formation of importin signaling complexes in injured axons. A gradient of nuclear RanGTP versus cytoplasmic RanGDP is thought to be fundamental for the organization of eukaryotic cells. Surprisingly, we find RanGTP in sciatic nerve axoplasm, distant from neuronal cell bodies and nuclei, and in association with dynein and importin-alpha. Following injury, localized translation of RanBP1 stimulates RanGTP dissociation from importins and subsequent hydrolysis, thereby allowing binding of newly synthesized importin-beta to importin-alpha and dynein. Perturbation of RanGTP hydrolysis or RanBP1 blockade at axonal injury sites reduces the neuronal conditioning lesion response. Thus, neurons employ localized mechanisms of Ran regulation to control retrograde injury signaling in peripheral nerve.     

瑞舒伐他汀促进缺氧复氧间充质干细胞增殖的机制研究

目的：探讨瑞舒伐他汀对缺氧复氧损伤后脂肪来源间充质干细胞增殖的影响及机制。方法：酶消化法分离小鼠的脂肪间充质干细胞（AD-MSCs）,流式细胞术检测CD90、CD44、CD34、CD45等细胞标志物。建立缺氧（H）6h/复氧（R）42h细胞模型,AD-MSCs分为3组：①对照组;②缺氧/复氧组（H/R）;③H/R＋瑞舒伐他汀干预组（浓度分别为10-8、10-7、10-6mol/L）。MTT法测定各组细胞增殖,免疫印迹法检测细胞内Akt、Erk及其磷酸化的表达水平。结果：流式细胞术结果显示脂肪间充质干细胞CD44及CD90阳性,CD34、CD45阴性。MTT实验显示在缺氧环境中,瑞舒伐他汀的干预可显著增加AD-MSCs的增殖（P〈0.05）。Westernblot检测pAkt及pErk的表达在瑞舒伐他汀干预组明显高于对照组和H/R组。（P〈0.05）。结论：瑞舒伐他汀可通过Akt、Erk信号途径促进H/R损伤后AD-MSCs的增殖。     

Phosphoflow-Based Evaluation of Mek Inhibitors as Small-Molecule Therapeutics for B-Cell Precursor Acute Lymphoblastic Leukemia

Upstream mutations that lead to constitutive activation of Erk in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are relatively common. In the era of personalized medicine, flow cytometry could be used as a rapid method for selection of optimal therapies, which may include drugs that target the Erk pathway. Here, we evaluated the utility of phospho-flow, compared to Western blotting, to monitor Erk pathway activation and its inhibition by targeted Mek kinase inhibitors in human BCP ALL. Because the Erk pathway is not only activated endogenously, by mutations, but also by normal extracellular stimulation through stromal contact and serum growth factors, we compared Erk activation ex vivo in ALL cells in the presence and absence of stroma and serum. Phospho-flow was able to readily detect changes in the pool of pErk1/2 that had been generated by normal microenvironmental stimuli in patient-derived BCP-ALL cells passaged in NSG mice, in viably frozen primary patient samples, and in fresh patient samples. Treatment with the Mek1/2 inhibitor selumetinib resulted in a rapid, complete and persistent reduction of microenvironment-generated pErk1/2. Imaging flow cytometry confirmed reduction of nuclear pErk1/2 upon selumetinib treatment. An ALL relapsing with an activating KRasG12V mutation contained higher endogenous as well as serum/stromal-stimulated levels of pErk1/2 than the matched diagnosis sample which lacked the mutation, but selumetinib treatment reduced pErk1/2 to the same level in both samples. Selumetinib and trametinib as Mek inhibitors were mainly cytostatic, but combined treatment with the PI3K∂ inhibitor CAL101 increased cytotoxicity. Thus phospho-flow cytometry could be used as a platform for rapid, individualized in vitro drug sensitivity assessment for leukemia patients at the time of diagnosis.     

Analysis of activated EGFR signalling pathways and their relation to laminin-5 γ2 chain expression in oral squamous cell carcinoma (OSCC)

Overexpression of epidermal growth factor receptor (EGFR) was shown for the majority of squamous cell carcinomas. The EGFR expression correlates to tumour size, stage and cytoplasmic accumulation of the laminin-5 γ2 chain (Ln-5/γ2), which is known as a marker of invading tumour cells. There is only limited knowledge if and how EGFR signalling pathways are important for invasion-associated processes and for the regulation of Ln-5/γ2. Therefore the distribution of phosphorylated Erk1/2, p38 MAPK and Akt was immunohistochemically defined in oral squamous cell carcinoma (OSCC) of different histological grade and compared to histological criteria of invasion and cytoplasmic Ln-5/γ2 deposition. With raising histological grade, there is a slight increase in nuclear pErk1/2-stained tumour cells (P=0.398) and a loss of nuclear (P=0.593) and increased cytoplasmic staining (P=0.144) of pAkt mainly in invading OSCC cells. Nuclear pp38 MAPK could only be sporadically detected in few cases. In case of pErk1/2 and pAkt, only a partial co-localisation could be revealed in cases with abundant kinases and Ln-5/γ2. Among the investigated kinases, only pAkt shows a relation to histological grade and invasion in OSCC. pErk1/2, pp38 MAPK and pAkt do not represent a direct link between EGFR and Ln-5 synthesis. Therefore, enhanced Ln-5/γ2 may be a secondary phenomenon of EGFR-induced tumour cell proliferation and dissemination.     

Extracellular signal-regulated kinase 1/2 mediates survival, but not axon regeneration, of adult injured central nervous system neurons in vivo

Neurotrophins play important roles in the response of adult neurons to injury. The intracellular signaling mechanisms used by neurotrophins to regulate survival and axon growth in the mature CNS in vivo are not well understood. The goal of this study was to define the role of the extracellular signal-regulated kinases 1/2 (Erk1/2) pathway in the survival and axon regeneration of adult rat retinal ganglion cells (RGCs), a prototypical central neuron population. We used recombinant adeno-associated virus (AAV) to selectively transduce RGCs with genes encoding constitutively active or wild-type mitogen-activated protein kinase kinase 1 (MEK1), the upstream activator of Erk1/2. In combination with anterograde and retrograde tracing techniques, we monitored neuronal survival and axon regeneration in vivo. MEK1 gene delivery led to robust and selective transgene expression in multiple RGC compartments including cell bodies, dendrites, axons and targets in the brain. Furthermore, MEK1 activation induced in vivo phosphorylation of Erk1/2 in RGC bodies and axons. Quantitative analysis of cell survival demonstrated that Erk1/2 activation promoted robust RGC neuroprotection after optic nerve injury. In contrast, stimulation of the Erk1/2 pathway was not sufficient to induce RGC axon growth beyond the lesion site. We conclude that the Erk1/2 pathway plays a key role in the survival of axotomized mammalian RGCs in vivo, and that activation of other signaling components is required for axon regeneration in the growth inhibitory CNS environment.     

TGF-beta activates Erk MAP kinase signalling through direct phosphorylation of ShcA

Erk1/Erk2 MAP kinases are key regulators of cell behaviour and their activation is generally associated with tyrosine kinase signalling. However, TGF-beta stimulation also activates Erk MAP kinases through an undefined mechanism, albeit to a much lower level than receptor tyrosine kinase stimulation. We report that upon TGF-beta stimulation, the activated TGF-beta type I receptor (TbetaRI) recruits and directly phosphorylates ShcA proteins on tyrosine and serine. This dual phosphorylation results from an intrinsic TbetaRI tyrosine kinase activity that complements its well-defined serine-threonine kinase function. TGF-beta-induced ShcA phosphorylation induces ShcA association with Grb2 and Sos, thereby initiating the well-characterised pathway linking receptor tyrosine kinases with Erk MAP kinases. We also found that TbetaRI is tyrosine phosphorylated in response to TGF-beta. Thus, TbetaRI, like the TGF-beta type II receptor, is a dual-specificity kinase. Recruitment of tyrosine kinase signalling pathways may account for aspects of TGF-beta biology that are independent of Smad signalling.     

Activation of Erk1/2 and Akt in astrocytes under ischemia

Substantial evidence has shown that extracellular signal-regulated kinases 1 and 2 (Erk1/2) and serine/threonine kinase (Akt) play important roles in regulating cell survival. We examined the activities of these kinases in astrocytes under ischemia in an anaerobic chamber. The level of phosphorylated Erk1/2 in astrocytes began to increase after 1 h ischemia, reached a maximum after 4 h ischemia, before decreasing from 5 to 6 h. Akt was activated later than Erk1/2. It was significantly increased after 4 h ischemia before declining steadily afterwards. Lactate dehydrogenase (LDH) assay and Hoechst nucleic staining indicated that U0126, which inhibits Erk1/2 phosphorylation, enhanced ischemia-induced cell death, whereas LY294002, which inhibits Akt phosphorylation, delayed cell death. These effects were dose-dependent. At 4 and 6 h ischemia, U0126-treated astrocytes expressed a lower level of Bcl-2 than controls. In contrast, LY294002-treated astrocytes expressed a higher level of Bcl-2 than controls as shown by Western blots. Bcl-x(L) expression level was not affected by either treatment. These data suggest that activation of the MAPK/Erk1/2 pathway might protect astrocytes from ischemic injury, but activation of the PI3-K/Akt pathway does not. The effect may involve Bcl-2 but not Bcl-x(L) expression.     

Soluble Axoplasm Enriched from Injured CNS Axons Reveals the Early Modulation of the Actin Cytoskeleton

Axon injury and degeneration is a common consequence of diverse neurological conditions including multiple sclerosis, traumatic brain injury and spinal cord injury. The molecular events underlying axon degeneration are poorly understood. We have developed a novel method to enrich for axoplasm from rodent optic nerve and characterised the early events in Wallerian degeneration using an unbiased proteomics screen. Our detergent-free method draws axoplasm into a dehydrated hydrogel of the polymer poly(2-hydroxyethyl methacrylate), which is then recovered using centrifugation. This technique is able to recover axonal proteins and significantly deplete glial contamination as confirmed by immunoblotting. We have used iTRAQ to compare axoplasm-enriched samples from naïve vs injured optic nerves, which has revealed a pronounced modulation of proteins associated with the actin cytoskeleton. To confirm the modulation of the actin cytoskeleton in injured axons we focused on the RhoA pathway. Western blotting revealed an augmentation of RhoA and phosphorylated cofilin in axoplasm-enriched samples from injured optic nerve. To investigate the localisation of these components of the RhoA pathway in injured axons we transected axons of primary hippocampal neurons in vitro. We observed an early modulation of filamentous actin with a concomitant redistribution of phosphorylated cofilin in injured axons. At later time-points, RhoA is found to accumulate in axonal swellings and also colocalises with filamentous actin. The actin cytoskeleton is a known sensor of cell viability across multiple eukaryotes, and our results suggest a similar role for the actin cytoskeleton following axon injury. In agreement with other reports, our data also highlights the role of the RhoA pathway in axon degeneration. These findings highlight a previously unexplored area of axon biology, which may open novel avenues to prevent axon degeneration. Our method for isolating CNS axoplasm also represents a new tool to study axon biology.     

Abrogation of TGFbeta signaling induces apoptosis through the modulation of MAP kinase pathways in breast cancer cells

Transforming growth factor beta (TGFbeta) can modulate the activity of various MAP kinases. However, how this pathway may mediate TGFbeta-induced malignant phenotypes remains elusive. We investigated the role of autocrine TGFbeta signaling through MAP kinases in the regulation of cell survival in breast carcinoma MCF-7 cells and untransformed human mammary epithelial cells (HMECs). Our results show that abrogation of autocrine TGFbeta signaling with the expression of a dominant negative type II TGFbeta receptor (DNRII) or the treatment with a TGFbeta type I receptor inhibitor significantly increased apoptosis in MCF-7 cell, but not in HMEC. The expression of DNRII markedly decreased activated/phosphorylated Erk, whereas increased activated/phosphorylated p38 in MCF-7 cells. In contrast, there was no or little change of phosphorylated Erk and p38 in HMECs after the expression of DNRII. Inhibition of Erk activity in MCF-7 control cell induced apoptosis whereas restoration of Erk activity in MCF-7 DNRII cell reduced apoptosis. Similarly, inhibition of p38 activity also inhibited apoptosis in MCF-7 DNRII cell. Thus, autocrine TGFbeta signaling can enhance the survival of MCF-7 cells by maintaining the level of active Erk high and the level of active p38 low. Furthermore, the survival properties of TGFbeta pathway appear related to transformation supporting the notion that it may be a potential target for cancer therapy.     

Squid neurofilaments. Phosphorylation and Ca2+-dependent proteolysis in situ.

Three major polypeptides co-purify with neurofilaments from squid (Loligo forbesi) axoplasm: P60 (apparent Mr 60,000), P200 (apparent Mr 200,000) and Band 1 (apparent Mr 400,000). Anti-IFA, a monoclonal antibody that recognizes an epitope common to all classes of intermediate filaments, binds to P200 and P60. When axoplasm is incubated with [32P]Pi, the major phosphorylated polypeptides are P200 and Band 1. We have investigated Ca2+-dependent proteolysis of [32P]phosphorylated axoplasm in order to localize the major sites of phosphorylation and to probe the arrangement of the polypeptides in the filament. The proteinase preferentially cleaves P200 and Band 1, liberating the phosphorylated domains. Analysis of proteolysed filaments by electron microscopy and gel electrophoresis shows that most of P200 and Band 1 can be cleaved while still maintaining intact filaments. We suggest that P200 is initially cleaved within a single highly sensitive region, generating two major fragments called P100p (apparent Mr 100,000) and P110s (apparent Mr 110,000). P100p contains the Anti-IFA epitope and co-sediments with filaments, whereas P110s is highly phosphorylated and does not sediment with filaments. Band 1 is cleaved to produce a soluble high-Mr fragment that is phosphorylated and that represents a major portion of the undigested component, whereas P60 is relatively resistant to limited proteolysis. Thus proteolysis appears to define two major filament domains: a conserved core that forms the backbone of the filament, and a highly phosphorylated peripheral region that is not essential for filament integrity.     

Topographic regulation of cytoskeletal protein phosphorylation by multimeric complexes in the squid giant fiber system

In mammalian and squid nervous systems, the phosphorylation of neurofilament proteins (NFs) seems to be topographically regulated. Although NFs and relevant kinases are synthesized in cell bodies, phosphorylation of NFs, particularly in the lys‐ser‐pro (KSP) repeats in NF‐M and NF‐H tail domains, seem to be restricted to axons. To explore the factors regulating the cellular compartmentalization of NF phosphorylation, we separated cell bodies (GFL) from axons in the squid stellate ganglion and compared the kinase activity in the respective lysates. Although total kinase activity was similar in each lysate, the profile of endogenous phosphorylated substrates was strikingly different. Neurofilament protein 220 (NF220), high‐molecular‐weight NF protein (HMW), and tubulin were the principal phosphorylated substrates in axoplasm, while tubulin was the principal GFL phosphorylated substrate, in addition to highly phosphorylated low‐molecular‐weight proteins. Western blot analysis showed that whereas both lysates contained similar kinases and cytoskeletal proteins, phosphorylated NF220 and HMW were completely absent from the GFL lysate. These differences were highlighted by P13^suc1 affinity chromatography, which revealed in axoplasm an active multimeric phosphorylation complex(es), enriched in cytoskeletal proteins and kinases; the equivalent P13 GFL complex exhibited six to 20 times less endogenous and exogenous phosphorylation activity, respectively, contained fewer cytoskeletal proteins and kinases, and expressed a qualitatively different cdc2‐like kinase epitope, 34 kDa rather than 49 kDa. Cell bodies and axons share a similar repertoire of molecular consitutents; however, the data suggest that the cytoskeletal/kinase phosphorylation complexes extracted from each cellular compartment by P13 are fundamentally different. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 89–102, 1999     

Human Nudel and NudE as regulators of cytoplasmic dynein in poleward protein transport along the mitotic spindle

Emerging evidence supports the idea that a signaling pathway containing orthologs of at least mammalian NudE and Nudel, Lis1, and cytoplasmic dynein is conserved for eukaryotic nuclear migration. In mammals, this pathway has profound impact on neuronal migration during development of the central nervous system. Lis1 and dynein are also involved in other cellular functions, such as mitosis. Here we show that Nudel also participates in a subset of dynein function in M phase. Nudel was specifically phosphorylated in M phase in its serine/threonine phosphorylation motifs, probably by Cdc2 and also Erk1 and -2. A fraction of Nudel bound to centrosomes strongly in interphase and localized to mitotic spindles in early M phase. By using mutants incapable of or simulating phosphorylation, we confirmed that phosphorylation of Nudel regulated the cell-cycle-dependent distribution, possibly by increasing its dissociation rate at the microtubule-organizing center. Moreover, phosphorylated Nudel or the phosphorylation-mimicking mutant bound Lis1 more efficiently. We further demonstrated that a Nudel mutant incapable of binding to Lis1 impaired the poleward movement of dynein and hence the dynein-mediated transport of kinetochore proteins to spindle poles along microtubules, a process contributing to inactivation of the spindle checkpoint in mitosis. These results point to the importance of Nudel-Lis1 interaction for the dynein activity in M phase and to a possible role of Nudel phosphorylation as facilitating such interaction. In addition, comparative studies suggest that NudE is also functionally related to its paralog, Nudel.     

The Raf-1 kinase associates with vimentin kinases and regulates the structure of vimentin filaments.

Using immobilized GST-Raf-1 as bait, we have isolated the intermediate filament protein vimentin as a Raf-1-associated protein. Vimentin coimmunoprecipitated and colocalized with Raf-1 in fibroblasts. Vimentin was not a Raf-1 substrate, but was phosphorylated by Raf-1-associated vimentin kinases. We provide evidence for at least two Raf-1-associated vimentin kinases and identified one as casein kinase 2. They are regulated by Raf-1, since the activation status of Raf-1 correlated with the phosphorylation of vimentin. Vimentin phosphorylation by Raf-1 preparations interfered with its polymerization in vitro. A subset of tryptic vimentin phosphopeptides induced by Raf-1 in vitro matched the vimentin phosphopeptides isolated from v-raf-transfected cells labeled with orthophosphoric acid, indicating that Raf-1 also induces vimentin phosphorylation in intact cells. In NIH 3T3 fibroblasts, the selective activation of an estrogen-regulated Raf-1 mutant induced a rearrangement and depolymerization of the reticular vimentin scaffold similar to the changes elicited by serum treatment. The rearrangement of the vimentin network occurred independently of the MEK/ERK pathway. These data identify a new branch point in Raf-1 signaling, which links Raf-1 to changes in the cytoskeletal architecture.     

Dynactin-dependent, dynein-driven vesicle transport in the absence of membrane proteins: a role for spectrin and acidic phospholipids

We reconstituted dynein-driven, dynactin-dependent vesicle transport using protein-free liposomes and soluble components from squid axoplasm. Dynein and dynactin, while necessary, are not the only essential cytosolic factors; axonal spectrin is also required. Spectrin is resident on axonal vesicles, and rebinds from cytosol to liposomes or proteolysed vesicles, concomitant with their dynein-dynactin-dependent motility. Binding of purified axonal spectrin to liposomes requires acidic phospholipids, as does motility. Using dominant negative spectrin polypeptides and a drug that releases PH domains from membranes, we show that spectrin is required for linking dynactin, and thereby dynein, to acidic phospholipids in the membrane. We verify this model in the context of liposomes, isolated axonal vesicles, and whole axoplasm. We conclude that spectrin has an essential role in retrograde axonal transport.     

A requirement for cytoplasmic dynein and dynactin in intermediate filament network assembly and organization

We present evidence that vimentin intermediate filament (IF) motility in vivo is associated with cytoplasmic dynein. Immunofluorescence reveals that subunits of dynein and dynactin are associated with all structural forms of vimentin in baby hamster kidney-21 cells. This relationship is also supported by the presence of numerous components of dynein and dynactin in IF-enriched cytoskeletal preparations. Overexpression of dynamitin biases IF motility toward the cell surface, leading to a perinuclear clearance of IFs and their redistribution to the cell surface. IF-enriched cytoskeletal preparations from dynamitin-overexpressing cells contain decreased amounts of dynein, actin-related protein-1, and p150Glued relative to controls. In contrast, the amount of dynamitin is unaltered in these preparations, indicating that it is involved in linking vimentin cargo to dynactin. The results demonstrate that dynein and dynactin are required for the normal organization of vimentin IF networks in vivo. These results together with those of previous studies also suggest that a balance among the microtubule (MT) minus and plus end-directed motors, cytoplasmic dynein, and kinesin are required for the assembly and maintenance of type III IF networks in interphase cells. Furthermore, these motors are to a large extent responsible for the long recognized relationships between vimentin IFs and MTs.     

Erk1/2-dependent phosphorylation of Galpha-interacting protein stimulates its GTPase accelerating activity and autophagy in human colon cancer cells

Galpha-interacting protein (GAIP) is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by the alpha-subunit of the trimeric G(i3) protein. Both proteins are part of a signaling pathway that controls lysosomal-autophagic catabolism in human colon cancer HT-29 cells. Here we show that GAIP is phosphorylated by an extracellular signal-regulated (Erk1/2) MAP kinase-dependent pathway sensitive to amino acids, MEK1/2 (PD098059), and protein kinase C (GF109203X) inhibitors. An in vitro phosphorylation assay demonstrates that Erk2-dependent phosphorylation of GAIP stimulates its GTPase-activating protein activity toward the Galpha(i3) protein (k = 0.187 +/- 0.001 s(-)(1), EC(50) = 1.12 +/- 0.10 microm) when compared with unphosphorylated recombinant GAIP (k = 0.145 +/- 0.003 s(-)(1), EC(50) = 3.16 +/- 0. 12 microm) or to GAIP phosphorylated by other Ser/Thr protein kinases (protein kinase C, casein kinase II). This stimulation and the phosphorylation of GAIP by Erk2 were abrogated when serine at position 151 in the RGS domain was substituted by an alanine residue using site-directed mutagenesis. Furthermore, the lysosomal-autophagic pathway was not stimulated in S151A-GAIP mutant-expressing cells when compared with wild-type GAIP-expressing cells. These results demonstrate that the GTPase-activating protein activity of GAIP is stimulated by Erk2 phosphorylation. They also suggested that Erk1/2 and GAIP are engaged in the signaling control of a major catabolic pathway in intestinal derived cells.