|
Article |
Address correspondence to Daniel B. Rifkin, Dept. of Cell Biology, New York University School of Medicine, 550 First Ave., New York, NY 10016. Tel.: (212) 263-5109. Fax: (212) 263-0595. email: rifkid01{at}med.nyu.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: LTBP; TGF-ß; integrin; vß6; latent TGF-ß
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The TGF-ßs are multipotent cytokines that modulate cell growth, inflammation, matrix synthesis, and apoptosis (Taipale et al., 1998). Defects in TGF-ß function are associated with a number of pathological states including tumor cell growth, fibrosis, emphysema, and autoimmune disease (Blobe et al., 2000; Morris et al., 2003). All three mammalian TGF-ß isoforms: TGF-ß1, 2, and 3 are synthesized as homodimeric proproteins (proTGF-ß). The dimeric propeptides, also known as the latency-associated protein (LAP), are cleaved from the mature TGF-ß dimer by furin-type enzymes but remain noncovalently associated with the mature cytokine (Dubois et al., 1995). The association between the TGF-ß1, 2, and 3 prodomains (LAPs) and the corresponding mature growth factors prevents signaling through the TGF-ß high affinity receptors (Lawrence et al., 1984). Thus, TGF-ß bioactivity requires dissociation from LAP, a process termed latent TGF-ß activation.
Early in the assembly of the TGF-ß latent complex, disulfide linkages are formed between cysteine(s) of LAP and specific cysteines in the LTBP (Miyazono et al., 1991; Gleizes et al., 1996; Saharinen et al., 1996). The ternary complex of TGF-ß, LAP, and LTBP is called the large latent complex (LLC; Fig. 1 A). Under most conditions, TGF-ß is secreted as part of the LLC (Taipale et al., 1998), which is consistent with the observation that LTBP-1 and TGF-ß1 expression are coregulated (Miyazono et al., 1991; Koski et al., 1999). The release of LAPTGF-ß, the small latent complex (SLC), without LTBP has been reported previously (Dallas et al., 1994), but this complex is inefficiently secreted (Miyazono et al., 1991). The biologic significance of these two different forms, LLC and SLC, of latent TGF-ß is unclear.
|
The integrin vß6 is an in vivo activator of latent TGF-ß1 and 3 (Munger et al., 1999; Annes et al., 2002). The expression of
vß6 is restricted to epithelia, and in most epithelia the integrin is normally expressed at low levels (Breuss et al., 1993). Mice lacking the ß6 subunit have persistent lung and skin inflammation but do not develop pulmonary fibrosis even when challenged with the profibrotic agent bleomycin (Huang et al., 1996; Munger et al., 1999). Analysis of gene expression in the lungs of bleomycin-treated mice suggests that lack of fibrosis in the ß6-null mice is a consequence of decreased TGF-ß activity because the overwhelming majority of TGF-ß responsive genes up-regulated in control mice are not increased in the ß6-null animals (Kaminski et al., 2000). The mechanism of integrin-mediated latent TGF-ß activation is not well understood. However, a direct interaction between
vß6 and the RGD amino acid sequence present in LAP is required, as mutation of this sequence to RGE eliminates activation. Importantly, integrin binding by itself to latent TGF-ß is not sufficient to activate TGF-ß as neither soluble
vß6, LAP-ß1binding to integrins
Vß1,
vß3 (Munger et al., 1998), nor
8ß1 (Lu et al., 2002) activates latent TGF-ß1.
Characterization of the mechanisms controlling the liberation of TGF-ß from its latent complex is central to understanding TGF-ß action. Although several activators (proteases, TSP-1, integrins) of latent TGF-ß have been identified, the molecular basis for TGF-ß activation remains only partially understood (Munger et al., 1997; Annes et al., 2003). For instance, there is strong evidence that LTBP plays an important role in protease-mediated latent TGF-ß activation as both inhibitors of tTGase (Kojima and Rifkin, 1993) and antibodies raised against LTBP-1 block activation of latent TGF-ß in several settings (Flaumenhaft et al., 1993; Nakajima et al., 1997; Gualandris et al., 2000). However, the function of LTBP in these systems is unclear. Unlike protease-mediated latent TGF-ß activation, vß6-mediated latent TGF-ß activation was reported not to require LTBP (Munger et al., 1999) based upon failure of anti-LTBP antibodies to block
vß6-mediated activation. However, it is possible that the roles of LTBP in protease- and integrin-mediated activation are distinct and cannot be inhibited in the same way. An important next step in understanding latent TGF-ß activation is to elucidate the function(s) of LTBP. Here, we present experimental results that define domains of LTBP-1 that are required for integrin-mediated latent TGF-ß activation, demonstrate an isoform (LTBP-1) specific function and gain insight into the mechanism of LTBP-1 regulation of TGF-ß generation.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An LTBP-1derived minimal TGF-ßbinding construct does not support Vß6-mediated activation of latent TGF-ß
A covalent interaction between LTBP and LAP might be required for Vß6-mediated activation because binding to LTBP may alter the structure of the SLC in a way that permits integrin-mediated activation, whereas in the absence of LTBP the SLC complex adopts a conformation unsuitable for integrin-mediated activation. If this hypothesis is correct, the small region of LTBP that covalently binds LAP might be sufficient to support
Vß6-mediated latent TGF-ß activation. To test this supposition, we used an LTBP-1Sderived construct that encodes only the LAP binding domain of LTBP (CR3) plus the flanking EGF-like repeats (EGF13 and 14; ECR3E, Fig. 2 A; Gleizes et al., 1996). The EGF-like domains are not required for LAP binding but are included to enhance expression of the protein product. As a control, we generated a similar expression construct that encodes CR4, which does not bind LAP (Gleizes et al., 1996), plus its flanking EGF-like domains (EGF15 and 16; Fig. 2 A, ECR4E). The effect of these expression constructs on latent TGF-ß activation was tested by using CHOß6 cells transduced with ECR3E, ECR4E or empty virus (Fig. 3 A). In this experiment, the CHOß6 cells (as opposed to the TGF-ß1//ß6 cells in Fig. 1) activate their endogenously produced latent TGF-ß (Fig. 3 A). Thus, the background levels of active TGF-ß are higher than those observed in Fig. 1. Contrary to our hypothesis, CHOß6 cells that expressed ECR3E demonstrated decreased latent TGF-ß activation (
50%), rather than unchanged or increased latent TGF-ß activation compared with mock-transduced and ECR4E-transduced cells (Fig. 3 A). The empty vector and the ECR4E expression construct had similar effects on latent TGF-ß activation.
|
|
A fragment of the hinge domain rescues ECR3E function
Based upon the preceding results that (1) Vß6-mediated activation requires a covalent interaction between LTBP and latent TGF-ß and that (2) ECR3E is not sufficient to support
Vß6-mediated activation, we suspected that an additional domain of LTBP-1S is required for latent TGF-ß activation by this mechanism. To define the domain of LTBP-1 involved in the activation process by a gain of function rather than a loss of function method, we required an experimental system in which
Vß6-mediated activation of latent TGF-ß was ineffective unless rescued by the introduction of LTBP-1S. The development of such a system was hindered by endogenous LTBP expression in every cell line tested (unpublished data). However, we circumvented this problem by taking advantage of CHO-ß6 cells that stably express ECR3E. These cells have an impaired ability to activate latent TGF-ß due to a block in LLC formation (similar to Fig. 3 B). Therefore, by transiently transfecting LTBP-1S into CHO-ß6/ECR3E cells, we could restore LLC formation and
Vß6-mediated latent TGF-ß activation.
We mapped the LTBP-1S domains required for Vß6-mediated latent TGF-ß activation by testing expression constructs with increasing NH2-terminal deletions (Fig. 2 A, constructs IIVII). The initial results of these experiments indicated a requirement for a region between amino acids 216 and 342 (CR1 and EGF2; Fig. 4 A) based upon the fact that construct III supports integrin-mediated activation, but construct IV, which lacks CR1 and EGF2, does not. Further NH2-terminal deletion of LTBP-1 (constructs VI and VII) did not restore
Vß6-mediated activation of latent TGF-ß (Fig. 4 A and not depicted). We attempted to identify the minimal LTBP-1Sderived construct that sustained activation (Fig. 4 B) by fusing the NH2-terminal portion of LTBP-1S (construct VIII, amino acids 1529) to ECR3E. This construct retained the ability to bind latent TGF-ß (Fig. 2 B), although the LTBP lacked the long EGF-like stretch and the COOH-terminal portion of LTBP-1S. Surprisingly, this construct rescued latent TGF-ß activation (Fig. 4 B, construct VIII). Further NH2-terminal deletions of construct VIII (constructs IX, X, and XII) did not prevent latent TGF-ß activation (Fig. 4 B). Indeed, a short amino acid sequence (amino acids 402529) derived from the hinge domain, when fused to ECR3E, was sufficient to restore activation by CHO-ß6/ECR3E cells (Fig. 4 B, construct XII). The apparent discrepancy between the results of the NH2-terminal deletion study that implicates amino acids 216342 (CR1 and EGF2) and the minimal domain experiment that identifies amino acids 402529 (hinge) as necessary for latent TGF-ß activation raises the possibility that amino acids 216342 modulate the function of the hinge domain by interacting with a distal portion of LTBP-1S, although other explanations are possible.
|
Next, we sought to determine if regions of LTBP-1S other than the hinge domain could support integrin-mediated activation. To accomplish this, we tested a mutant form of LTBP-1 that lacked amino acids 402449 for the ability to support activation (Fig. 2 A, construct I). Indeed, construct I failed to support Vß6-mediated activation (Fig. 5 B). Interestingly, the hinge domain of LTBP-1 is not conserved among the other LTBP isoforms (Fig. 5 A). This fact raised the possibility that LTBP-1 performs a function that is not fulfilled by the other LTBP isoforms. To test this hypothesis, we transfected CHO-ECR3E cells with an LTBP-3 expression construct and measured latent TGF-ß activation (Fig. 5 B). Despite the fact that LTBP-3 was secreted in complex with latent TGF-ß at levels similar to LTBP-1 (Fig. 5 C, compare LTBP-1S with LTBP-3 [LTBP1 403-449]), LTBP-3 did not support latent TGF-ß activation (Fig. 5 B). As a further test of possible isoform specificity, an LTBP-3 expression construct in which the native hinge domain was replaced with that of LTBP-1 (LTBP-3 with LTBP-1 hinge) was tested for the ability to support
Vß6-mediated activation. Strikingly, LTBP-3 with LTBP-1 hinge supported TGF-ß generation (Fig. 5 B). This result indicates that the hinge domain of LTBP-1 confers an isoform-specific function within the LTBP family.
|
The cosegregation of two activities for amino acids 402449 of LTBP-1, namely support of latent TGF-ß activation and ECM association, led us to suspect that fixation of latent TGF-ß is required for Vß6-mediated latent TGF-ß activation. According to this hypothesis, expression of the minimal TGF-ß binding construct, ECR3E, blocks integrin-mediated activation by preventing LLC formation thereby disrupting localization of the latent TGF-ß complex. Therefore, we reasoned that artificial targeting of ECR3E-bound latent TGF-ß to the vicinity of the ECM should restore
Vß6-mediated latent TGF-ß activation. To test this prediction, we took advantage of the fact that the ECR3E constructs encode tandem COOH-terminal HA epitopes. Wells of a 96-well microtiter plate were either mock coated or anti-HA Ab coated (25 µg/ml). These wells were subsequently used to test latent TGF-ß activation by CHO K7 cells engineered to express (1) ß6 cDNA (Fig. 6 A, bars 1 and 5), (2) ECR3E and ß6 cDNAs (Fig. 6 A, bars 2 and 6), (3) ECR3E cDNA (Fig. 6 A, bars 3 and 7), or (4) empty vectors (Fig. 6 A, bars 4 and 8). The ß6-expressing cells activated their endogenous latent TGF-ß when plated on either surface (Fig. 6 A, bars 1 and 5). However, by comparing bars 2 and 6 in Fig. 6 A, it is clear that the CHO-ECR3E/ß6 cells demonstrate an enhanced (approximately four times) activation of latent TGF-ß on wells coated with anti-HA Ab compared with mock-coated wells. In fact, on mock coated wells CHO-ECR3E/ß6 activated approximately half as much latent TGF-ß as CHO-ß6 cells (Fig. 6 A, compare bar 1 with bar 2); on anti-HA Ab-coated wells CHO-ECR3E/ß6 cells activated approximately two times more latent TGF-ß than CHO-ß6 (Fig. 6 A, compare bar 5 with bar 6). Additional controls demonstrate that expression of both ECR3E and
Vß6 are required for enhanced activation on anti-HA Ab-coated wells (Fig. 6 A, bars 3, 4, 7, and 8). We also observed a direct relationship between the HA Ab-coating concentration and TGF-ß generation by CHO-ECR3E/ß6 cells (Fig. 6 B). In all cases, the generation of active TGF-ß was blocked by an
Vß6-specific blocking Ab (Fig. 6 B). Similar results were obtained with SW-480 cells (not depicted). These and the previous results suggest that LTBP-1Sderived expression constructs that support
Vß6-mediated activation do so because they effectively localize latent TGF-ß, whereas constructs that fail to support activation do so because they cannot associate with the ECM.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The requirement of LTBP-dependent localization of latent TGF-ß for activation suggests that TGF-ß activity is regulated not only by the expression of latent TGF-ß and an activator, but also by the different forms of LLC, the availability of a binding partner and the supply of ECM-stored latent TGF-ß. For instance, removal of the NH2-terminal region of LTBP with plasmin generates a form of LTBP that resembles platelet LTBP and fails to be incorporated into the ECM (Taipale et al., 1994a). Consistent with our hypothesis, expression constructs that encode forms of LLC similar to platelet LLC (Fig. 2 A, constructs V, VI, VII) result in protein products that fail to support activation in our system (Kanzaki et al., 1990; Miyazono et al., 1991). In addition, the various isoforms of LTBP may differentially regulate TGF-ß activity, as latent TGF-ß bound to LTBP-3 is not a substrate for Vß6-mediated activation. It is interesting that the greatest degree of sequence divergence between LTBP-1 and LTBP-3 is within their hinge domains and that it is this region of the molecule that confers an LTBP-1specific function. This specificity for LTBP-1 hinge domain in
Vß6-mediated activation of latent TGF-ß suggests that the hinge domains of LTBP-3, -2, and -4 may have distinct functions and cannot substitute for each other under all conditions. Furthermore, the activities of different LTBP isoforms and splice variants may relate to their distinct patterns of intra- and extracellular localization.
Our work indicates that processes that influence matrix deposition of latent TGF-ß are predicted to affect Vß6-dependent TGF-ß generation both positively and negatively. Interestingly, matrix association of latent TGF-ß may differentially influence protease-mediated activation versus
Vß6-mediated activation. Protease-mediated latent TGF-ß activation may require release of latent TGF-ß from the ECM for subsequent cell surface localization and activation (Flaumenhaft and Rifkin, 1992a; Nunes et al., 1997), whereas
Vß6-mediated latent TGF-ß activation may occur while latent TGF-ß is fixed. To better understand latent TGF-ß activation and TGF-ß activity in general, it will be important to answer questions such as: (1) Is LTBP required for other activation mechanisms? (2) What are the roles of the various LTBP isoforms in these processes? (3) How is activation of SLC accomplished/regulated?
Mapping identified a portion of the hinge domain, amino acids 402449, and the TGF-ßbinding domain as the minimal LTBP-1derived domains that support Vß6-mediated latent TGF-ß activation. (The relationship of the hinge domain and the tTGase cross-linking site is unclear.) This hinge region may function by targeting latent TGF-ß to an extracellular location. The hinge sequence of LTBP-1 contains a putative heparin-binding site that might facilitate ECM or cell surface targeting of the latent TGF-ß complex (Oklu et al., 1998; Dallas et al., 2002; Isogai et al., 2003). Currently, we are trying to identify the hinge domain binding partner.
To understand the mechanism of Vß6-mediated activation, it is necessary to appreciate the function of LTBP in this process. The results presented here suggest that LTBP promotes
Vß6-mediated activation by both concentrating and fixing the latent complex. The requirement for integrincytoskeleton interaction for latent TGF-ß activation suggests that force generation by the integrin may be necessary. Indeed, the mechanism by which integrins generate force may explain why fixation of latent TGF-ß in the ECM is important: integrin-dependent force generation across the cell membrane increases with increasing resistance (Choquet et al., 1997). We hypothesize that fixation of latent TGF-ß provides resistance to integrin pulling, allowing focal contact reinforcement and force generation sufficient to release TGF-ß from latency. Our current conception of
Vß6-mediated latent TGF-ß activation is represented schematically in Fig. 8. In this model, LTBP-1 but not LTBP-3 is able to fix the latent complex to the ECM. Consequently, the LTBP-1TGF-ß complex is incorporated into the ECM and, therefore, when bound by
Vß6, promotes focal adhesion formation, force generation, and release of TGF-ß from the latent complex. By contrast, integrin binding to latent the LTBP-3TGF-ß complex does not result in adhesion complex formation, force generation, and latent TGF-ß activation. Consistent with this model, we predict that protease-mediated release of a soluble latent TGF-ß restricts TGF-ß generation by the integrin
Vß6. Thus, the function of LTBP-1 in
Vß6-mediated latent TGF-ß activation is to concentrate and fix latent TGF-ß so that integrin pulling is opposed. This resistance alters the conformation of LAP and releases TGF-ß. The necessity of localization and force generation provide mechanisms for spatially restricting TGF-ß activity and raises the threshold of latent TGF-ß activation to limit inappropriate cytokine activity.
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells were cultured in Dulbecco's minimum essential medium (DMEM) containing 10% heat-inactivated FCS. Mouse mAb HA.11 against the HA epitope was purchased from Covance Research Products (Berkeley Antibody Company). VB3A9 is an mAb directed against the LAP portion of human TGF-ß1 (Munger et al., 1998). Ab39, rabbit polyclonal antisera raised against platelet LTBP-1, was provided by C.-H. Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden). Mouse anti-Vß6 mAb 10D5 (Huang et al., 1998) was a gift of D. Sheppard. Recombinant simian TGF-ß1 LAP was produced as described previously (Munger et al., 1998). All molecular biology enzymes were purchased from Roche Diagnostics. Recombinant human TGF-ß1 SLC was purchased from R&D Systems.
Constructs and vectors
pcDNA3 and pSecTag 2C vectors were obtained from Invitrogen. PMX retroviral vector was provided by T. Kitamura (Tokyo University, Tokyo, Japan). Simian TGF-ß1 cDNA was a gift from R. Derynck (UCSF). Human TGF-ß1 C33S cDNA was a gift from J. Keski-Oja (University of Helsinki, Helsinki, Finland). Human LTBP-1S cDNA (pSV7d-BP13) was a gift of K. Miyazono and C.-H. Heldin (Ludwig Institute for Cancer Research). ß6-Integrin cDNA was a gift of D. Sheppard.
Generation of pcDNA3-LTBP-1S and pcDNA3-N441 expression vectors was described previously (Nunes et al., 1997). In brief, pcDNA3-LTBP-1S was constructed by fusing the DraIDrI fragment of pSV7d-BP13 (nucleotides 684543) in frame with the baculovirus glycoprotein GP67 signal sequence in pcDNA3. Similarly, pcDNA3-
N441 was generated by fusing the HpaIDraI fragment (nucleotides 14144543) of pSV7d-BP13 in frame with the GP67 signal sequence in the pcDNA3 vector.
The PCR primers and templates used for generation of the expression constructs described below are given in Table I. The expression construct pcDNA3-ECR3E-2HA (nucleotides 28723408) was generated by PCR amplification (primers 1, 2) using pcDNA3-LTBP-1S as the template. This PCR product was fused to the BM40 signal sequence the pRcCMV/Ac7 vector (gift of R. Timpl, Max-Plank Institute, Martinsried, Germany). The pRcCMV/Ac7ECR3E vector was digested with HpaIXbaI and an adapter cassette coding for two copies of the HA-epitope was subcloned in frame and downstream of the ECR3E coding sequence. The ECR3E-2HA coding sequence was subsequently transferred from pRcCMV/Ac7 to an intermediate vector (pKN185; gift of Y. Yamada, National Institutes of Health) using an EagI digest of both vectors. The ECR3E-2HA coding sequence was subsequently released from PKN185-ECR3E-2HA by a HindIIIBamHI digest and subcloned into pCDNA3 (HindIIIBamHI) and pBluescript II KS+ (Strategene). ECR3E-2HA was transferred from pBluescript II KS+ -ECR3E-2HA (pBlue-ECR3E-2HA) to PMX (HindIIINotI digest). To make PMX-ECR4E-2HA, the ECR4E coding sequence was amplified (primers 3, 4) using pcDNA3-LTBP-1S as the template and subcloned into the pBlue-ECR3E-2HA vector backbone, which retained the BM40 signal sequence and the 2HA epitopes. This was accomplished by SpeIHpaI digest of both the ECR4E PCR product and pBlue-ECR3E-2HA vector and ligation of the digested ECR4E PCR product into the pBlue-ECR3E-2HA vector backbone. ECR4E-2HA was subsequently transferred to the PMX retroviral vector by HindIIINotI digest of pBlue-ECR4E-2HA, purification of the ECR4E-2HA insert, and ligation into similarly digested PMX retrovirus vector DNA.
|
A number of LTBP-1S deletion constructs were made in pSecTag2C using strand overlap extension PCR reactions pSecTag2C-N216/
C529-ECR3E-2HA (primers 24/25,11/12); pSecTag2C-
N342/
C529-ECR3E-2HA (primers 26/25, 11/12); pSecTag2C-
N402/
C529-ECR3E-2HA (primers 27/25, 11/12); pSecTag2C-
N449/
C529-ECR3E-2HA (primers 28/25, 11/12). In all cases, the amplified products of reaction 3 were NotIIXhoI digested before being subcloned into similarly digested pSecTag2C vector. pSecTag2C-
N402/
C449-ECR3E-HA L1
L2 was generated by two successive QuickChange Site-Directed Mutagenesis reactions (Stratagene). The first reaction deleted the phenylalanine and proline (amino acids 1060 and 1061 of LTBP-1S) that are found in CR3 of human LTBP-1 but not in CR3 of human LTBP-2. This was accomplished using a sense primer (5'-GGGGAGATAACTGCGAAATCTGCCCGGTCTTGGGAACTGC-3') and an equivalent antiparallel primer. The second reaction mutated the glutamate and isoleucine of human LTBP-1 CR3 (amino acids 1058 and 1059 of LTBP-1S) to aspartate and leucine, which are found in the homologous region of human CR3 of LTBP-2. This was accomplished using a sense primer (5'-GGGGAGATAACTGCGACCTCTGCCCGGTCTTGGGAACTGC-3') and an equivalent antiparallel primer. pSecTag2C
N402/
C449-ECR3E-HA was generated by PCR amplification (primers 29, 30). The PCR fragment was digested with HindIII and BamHI and cloned into a similarly digested pSecTag2C-ECR3E-HA vector. pSecTag2C-
N19/
C449-ECR3E-HA was constructed by PCR amplification (primers 31, 32). The PCR product was digested with HindIII and BamHI and cloned into similarly digested pSecTag2C-ECR3E-HA vector.
pcDNA-LTBP1 402-449 was constructed by PCR amplification (primers 33/34, 35/36) of 5' and 3' fragments that were digested (reaction 1, ClaIEcoRV; reaction 2, HpaIEcoRI), purified and cloned into pcDN3-LTBP-1S digested with ClaIEcoRI. HA tagged versions of pcDNA3-LTBP-1, -LTBP-1S-
N402-449, and -LTBP-
N402 were generated by digesting these constructs with EcoRIXbaI and cloning in a COOH-terminal HA tagged amplified product from pcDNA3-LTBP-1S (primers 37, 38). PMX-
N402/
C449-ECR3E-HA, PMX-
N402/
C449-ECR3E-HA L1
2, and PMX-
N449/
C529-ECR3E-HA were generated by PCR amplification using the respective pSecTag2C vectors as templates (primers 39, 40). The PCR products were digested with EcoRIXhoI and cloned into PMX vector digested with EcoRISalI. PMX-ß6 retroviral vector was prepared by isolating the Pme digestion fragment of pHygrobeta6 and cloning into filled-in EcoRI digested PMX virus (Annes et al., 2002). The LTBP-3 with the LTBP-1 hinge expression construct was synthesized by a series of strand-overlap extension PCR reactions that sewed the LTBP-1 hinge domain into the LTBP-3 coding sequence. First, overlapping PCR products derived from LTBP-3 (upstream of the hinge domain) and LTBP-1 were synthesized separately (primers 41/42 and 43/44). These products were then combined together for reaction 3. A fourth PCR reaction was set up to generate an LTBP-3derived product (downstream of the hinge domain) that also overlapped with the LTBP-1 hinge domain (primers 45/46). This product was combined with the product of reaction 3 and sewn together in a fifth PCR reaction (primers 41/46). The product of this PCR reaction was digested with EcoRV and Nru and cloned into similarly digested pcDNA3-LTBP-3. All constructs were checked by automated sequencing.
TGF-ß bioassays
TGF-ß activation was measured using CHO cells stably transfected with an ECR3E-2HA expression construct and the ß6-integrin subunit (CHO-ECR3E-2HA/ß6) subsequently transiently transfected with proTGF-ß1 and various LTBP-1Sderived cDNAs. Before transient transfection, test cells were plated at 4 x 105 cells per 35-mm well in DMEM/10% FCS. After 16 h, cells were transfected with the LTBP-1S expression constructs (1 µg per well) and the TGF-ß1 expression construct (400 ng per well) using LipofectAMINE Plus. After 16 h, the cells were collected in 3 ml of DMEM/10% FCS and replated in 96-well and 24-well plates (50 and 500 µl per well, respectively). TMLC (1.5 x 104) were added to the 96-well plates (final volume, 100 µl per well). When appropriate, 10D5 (20 µg/ml) or LAP (100 µg/ml) was added to the co-culture. Conditioned media were generated in the 24-well plate monocultures. After 1624 h, TGF-ß activation was assessed by measuring luciferase activity in the cell lysates from co-cultures (Abe et al., 1994). In addition, the conditioned media from monoculture wells was collected after 1624 h and analyzed by immunoblotting (Ab39 or HA.11) for secretion of the various LTBP forms as well as by TMLC assay for total TGF-ß secretion by heat activating latent TGF-ß (80°C for 10 min). The samples were incubated with TMLC overnight and luciferase activity measured.
To test TGF-ß1 SLC as a substrate for Vß6-mediated latent TGF-ß activation, TGF-ß1/ cells and TGF-ß1//ß6 cells (1.5 x 104 cells per well) were co-cultured with TMLC (1.0 x 104 cells per well) in 96-well plates in the presence of TGF-ß1 SLC (0200 ng/ml of TGF-ß1 SLC). After 1624 h, luciferase activity in the cell lysates was measured. To test if
Vß6-mediated latent TGF-ß activation requires TGF-ß binding to LTBP, TGF-ß1//ß6 cells (8 x 104 cells per 35-mm well) were transiently transfected with cDNAs encoding either wild-type human TGF-ß1 (400 ng per well) or human TGF-ß1C:S (400 ng per well). The establishment of co-cultures to measure TGF-ß activation and monocultures to measure total TGF-ß secretion was as described previously (Abe et al., 1994).
ECM deposition of latent TGF-ß was testing by using, CHO-K7, SW-480, or 2T3 osteoblast precursor cells transduced with PMX virus, 402-449ECR3E virus, or 450529ECR3E virus and subsequently transfected with human TGF-ß1 cDNA. 2 d after transfection, cells were replated in 96-well plates, 20,000 cells per well. 24 h later, cells in 96-well plates were washed once with PBS, and detached from the cell culture plate with 20 mM EDTA/PBS at 37°C for 3040 min. Wells were washed two more times with PBS, and 100 µl DMEM was added into each well. The 96-well plate was incubated at 80°C for 20 min to activate and release matrix-bound latent TGF-ß. The media were collected and put onto TMLC to measure TGF-ß activity.
To test the ability of CHO-ECR3E-2HA/ß6 cells to activate latent TGF-ß deposited in the ECM, CHO cells stably transfected with ECR3E (CHO-ECR3E) or LTBP-1S (CHO-LTBP) were allowed to synthesize a matrix in 96-wells (5.0 x 104 cells per well) for 48 h before they were removed with PBS/15 mM EDTA. CHO-ECR3E-2HA/ß6 cells (2.0 x 104 cells per well) or SW480-ECR3E-2HA/ß6 cells (2.5 x 104 cells per well) were plated on the preformed matrices with reporter cells (1.5 x 104 cells per well; 100 µl final volume) to measure TGF-ß activity. A TGF-ß neutralizing antibody (1D11; 15 µg/ml) or an Vß6-blocking antibody (10D5; 20 µg/ml) was added to co-culture wells as appropriate.
To test if artificially targeting ECR3E-2HAbound latent TGF-ß to the vicinity of the ECM restored latent TGF-ß activation, the wells of a 96-well plate were either coated with the mouse mAb HA.11 (25 µg/ml) in PBS or mock coated for 1.5 h at 37°C. The wells were washed with DMEM/10% FCS, and various cell types (SW480/ß6, SW480-ECR3E-2HA/ß6, SW480-ECR3E-2HA, SW480-PMX) were added (2.0 x 104 cells per well) to both anti-HAcoated and mock-coated wells. TMLC (1.5 x 104 cells per well) were added to each well to a final volume of 100 µl per well. TGF-ß neutralizing antibody (1D11; 15 µg/ml) was added as appropriate. After 1624 h, luciferase activity was assayed.
![]() |
Acknowledgments |
---|
This work was supported by grants CA34282, CA78422, DE-13742 (to D.B. Rifkin), and T32 GM07308 (to J.P. Annes).
Submitted: 6 January 2004
Accepted: 30 April 2004
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abe, M., J.G. Harpel, C.N. Metz, I. Nunes, D.J. Loskutoff, and D.B. Rifkin. 1994. An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. Anal. Biochem. 216:276284.[CrossRef][Medline]
Abe, M., N. Oda, and Y. Sato. 1998. Cell-associated activation of latent transforming growth factor-beta by calpain. J. Cell. Physiol. 174:186193.[CrossRef][Medline]
Annes, J.P., J.S. Munger, and D.B. Rifkin. 2003. Making sense of latent TGFbeta activation. J. Cell Sci. 116:217224.
Annes, J.P., D.B. Rifkin, and J.S. Munger. 2002. The integrin alphaVbeta6 binds and activates latent TGFbeta3. FEBS Lett. 511:6568.[CrossRef][Medline]
Blobe, G.C., W.P. Schiemann, and H.F. Lodish. 2000. Role of transforming growth factor beta in human disease. N. Engl. J. Med. 342:13501358.
Breuss, J.M., N. Gillett, L. Lu, D. Sheppard, and R. Pytela. 1993. Restricted distribution of integrin beta 6 mRNA in primate epithelial tissues. J. Histochem. Cytochem. 41:15211527.
Choquet, D., D.P. Felsenfeld, and M.P. Sheetz. 1997. Extracellular matrix rigidity causes strengthening of integrin-cytoskeleton linkages. Cell. 88:3948.[Medline]
Dallas, S.L., D.R. Keene, S.P. Bruder, J. Saharinen, L.Y. Sakai, G.R. Mundy, and L.F. Bonewald. 2000. Role of the latent transforming growth factor beta binding protein 1 in fibrillin-containing microfibrils in bone cells in vitro and in vivo. J. Bone Miner. Res. 15:6881.[Medline]
Dallas, S.L., S. Park-Snyder, K. Miyazono, D. Twardzik, G.R. Mundy, and L.F. Bonewald. 1994. Characterization and autoregulation of latent transforming growth factor beta (TGF beta) complexes in osteoblast-like cell lines. Production of a latent complex lacking the latent TGF beta-binding protein. J. Biol. Chem. 269:68156821.
Dallas, S.L., J.L. Rosser, G.R. Mundy, and L.F. Bonewald. 2002. Proteolysis of latent transforming growth factor-beta (TGF-beta)-binding protein-1 by osteoclasts. A cellular mechanism for release of TGF-beta from bone matrix. J. Biol. Chem. 277:2135221360.
Dubois, C.M., M.H. Laprise, F. Blanchette, L.E. Gentry, and R. Leduc. 1995. Processing of transforming growth factor beta 1 precursor by human furin convertase. J. Biol. Chem. 270:1061810624.
Flaumenhaft, R., M. Abe, Y. Sato, K. Miyazono, J. Harpel, C.H. Heldin, and D.B. Rifkin. 1993. Role of the latent TGF-beta binding protein in the activation of latent TGF-beta by co-cultures of endothelial and smooth muscle cells. J. Cell Biol. 120:9951002.[Abstract]
Flaumenhaft, R., and D.B. Rifkin. 1992a. Cell density dependent effects of TGF-beta demonstrated by a plasminogen activator-based assay for TGF-beta. J. Cell. Physiol. 152:4855.[Medline]
Flaumenhaft, R., and D.B. Rifkin. 1992b. The extracellular regulation of growth factor action. Mol. Biol. Cell. 3:10571065.[Medline]
Gleizes, P.E., R.C. Beavis, R. Mazzieri, B. Shen, and D.B. Rifkin. 1996. Identification and characterization of an eight-cysteine repeat of the latent transforming growth factor-beta binding protein-1 that mediates bonding to the latent transforming growth factor-beta1. J. Biol. Chem. 271:2989129896.
Gualandris, A., J.P. Annes, M. Arese, I. Noguera, V. Jurukovski, and D.B. Rifkin. 2000. The latent transforming growth factor-beta-binding protein-1 promotes In vitro differentiation of embryonic stem cells into endothelium. Mol. Biol. Cell. 11:42954308.
Huang, X., J. Wu, S. Spong, and D. Sheppard. 1998. The integrin alphavbeta6 is critical for keratinocyte migration on both its known ligand, fibronectin, and on vitronectin. J. Cell Sci. 111:21892195.
Huang, X.Z., J.F. Wu, D. Cass, D.J. Erle, D. Corry, S.G. Young, R.V. Farese, and D. Sheppard. 1996. Inactivation of the integrin beta 6 subunit gene reveals a role of epithelial integrins in regulating inflammation in the lung and skin. J. Cell Biol. 133:921928.[Abstract]
Isogai, Z., R.N. Ono, S. Ushiro, D.R. Keene, Y. Chen, R. Mazzieri, N.L. Charbonneau, D.P. Reinhardt, D.B. Rifkin, and L.Y. Sakai. 2003. Latent transforming growth factor beta-binding protein 1 interacts with fibrillin and is a microfibril-associated protein. J. Biol. Chem. 278:27502757.
Kaminski, N., J.D. Allard, J.F. Pittet, F. Zuo, M.J. Griffiths, D. Morris, X. Huang, D. Sheppard, and R.A. Heller. 2000. Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis. Proc. Natl. Acad. Sci. USA. 97:17781783.
Kanzaki, T., A. Olofsson, A. Moren, C. Wernstedt, U. Hellman, K. Miyazono, L. Claesson-Welsh, and C.H. Heldin. 1990. TGF-beta 1 binding protein: a component of the large latent complex of TGF-beta 1 with multiple repeat sequences. Cell. 61:10511061.[Medline]
Kojima, S., and D.B. Rifkin. 1993. Mechanism of retinoid-induced activation of latent transforming growth factor-beta in bovine endothelial cells. J. Cell. Physiol. 155:323332.[Medline]
Koski, C., J. Saharinen, and J. Keski-Oja. 1999. Independent promoters regulate the expression of two amino terminally distinct forms of latent transforming growth factor-beta binding protein-1 (LTBP-1) in a cell type-specific manner. J. Biol. Chem. 274:3261932630.
Lack, J., J.M. O'Leary, V. Knott, X. Yuan, D.B. Rifkin, P.A. Handford, and A.K. Downing. 2003. Solution structure of the third TB domain from LTBP1 provides insight into assembly of the large latent complex that sequesters latent TGF-beta. J. Mol. Biol. 334:281291.[CrossRef][Medline]
Lawrence, D.A., R. Pircher, C. Kryceve-Martinerie, and P. Jullien. 1984. Normal embryo fibroblasts release transforming growth factors in a latent form. J. Cell. Physiol. 121:184188.[Medline]
Lu, M., J.S. Munger, M. Steadele, C. Busald, M. Tellier, and L.M. Schnapp. 2002. Integrin alpha8beta1 mediates adhesion to LAP-TGFbeta1. J. Cell Sci. 115:46414648.
Miyazono, K., A. Olofsson, P. Colosetti, and C.H. Heldin. 1991. A role of the latent TGF-beta 1-binding protein in the assembly and secretion of TGF-beta 1. EMBO J. 10:10911101.[Abstract]
Morris, D.G., X. Huang, N. Kaminski, Y. Wang, S.D. Shapiro, G. Dolganov, A. Glick, and D. Sheppard. 2003. Loss of integrin alpha(v)beta6-mediated TGF-beta activation causes Mmp12-dependent emphysema. Nature. 422:169173.[CrossRef][Medline]
Munger, J.S., J.G. Harpel, P.E. Gleizes, R. Mazzieri, I. Nunes, and D.B. Rifkin. 1997. Latent transforming growth factor-beta: structural features and mechanisms of activation. Kidney Int. 51:13761382.[Medline]
Munger, J.S., J.G. Harpel, F.G. Giancotti, and D.B. Rifkin. 1998. Interactions between growth factors and integrins: latent forms of transforming growth factor-beta are ligands for the integrin alphavbeta1. Mol. Biol. Cell. 9:26272638.
Munger, J.S., X. Huang, H. Kawakatsu, M.J. Griffiths, S.L. Dalton, J. Wu, J.F. Pittet, N. Kaminski, C. Garat, M.A. Matthay, et al. 1999. The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell. 96:319328.[Medline]
Nakajima, Y., K. Miyazono, M. Kato, M. Takase, T. Yamagishi, and H. Nakamura. 1997. Extracellular fibrillar structure of latent TGF beta binding protein-1: role in TGF beta-dependent endothelial-mesenchymal transformation during endocardial cushion tissue formation in mouse embryonic heart. J. Cell Biol. 136:193204.
Nunes, I., P.E. Gleizes, C.N. Metz, and D.B. Rifkin. 1997. Latent transforming growth factor-beta binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-beta. J. Cell Biol. 136:11511163.
Oklu, R., J.C. Metcalfe, T.R. Hesketh, and P.R. Kemp. 1998. Loss of a consensus heparin binding site by alternative splicing of latent transforming growth factor-beta binding protein-1. FEBS Lett. 425:281285.[CrossRef][Medline]
Ramirez, F., and L. Pereira. 1999. The fibrillins. Int. J. Biochem. Cell Biol. 31:255259.[CrossRef][Medline]
Saharinen, J., and J. Keski-Oja. 2000. Specific sequence motif of 8-Cys repeats of TGF-beta binding proteins, LTBPs, creates a hydrophobic interaction surface for binding of small latent TGF-beta. Mol. Biol. Cell. 11:26912704.
Saharinen, J., J. Taipale, and J. Keski-Oja. 1996. Association of the small latent transforming growth factor-beta with an eight cysteine repeat of its binding protein LTBP-1. EMBO J. 15:245253.[Abstract]
Sinha, S., C. Nevett, C.A. Shuttleworth, and C.M. Kielty. 1998. Cellular and extracellular biology of the latent transforming growth factor-beta binding proteins. Matrix Biol. 17:529545.[CrossRef][Medline]
Taipale, J., and J. Keski-Oja. 1997. Growth factors in the extracellular matrix. FASEB J. 11:5159.
Taipale, J., K. Koli, and J. Keski-Oja. 1992. Release of transforming growth factor-beta 1 from the pericellular matrix of cultured fibroblasts and fibrosarcoma cells by plasmin and thrombin. J. Biol. Chem. 267:2537825384.
Taipale, J., S. Matikainen, M. Hurme, and J. Keski-Oja. 1994a. Induction of transforming growth factor beta 1 and its receptor expression during myeloid leukemia cell differentiation. Cell Growth Differ. 5:13091319.[Abstract]
Taipale, J., K. Miyazono, C.H. Heldin, and J. Keski-Oja. 1994b. Latent transforming growth factor-beta 1 associates to fibroblast extracellular matrix via latent TGF-beta binding protein. J. Cell Biol. 124:171181.[Abstract]
Taipale, J., J. Saharinen, and J. Keski-Oja. 1998. Extracellular matrix-associated transforming growth factor-beta: role in cancer cell growth and invasion. Adv. Cancer Res. 75:87134.[Medline]
Tsuji, T., F. Okada, K. Yamaguchi, and T. Nakamura. 1990. Molecular cloning of the large subunit of transforming growth factor type beta masking protein and expression of the mRNA in various rat tissues. Proc. Natl. Acad. Sci. USA. 87:88358839.[Abstract]
Unsold, C., M. Hyytiainen, L. Bruckner-Tuderman, and J. Keski-Oja. 2001. Latent TGF-beta binding protein LTBP-1 contains three potential extracellular matrix interacting domains. J. Cell Sci. 114:187197.
Weinacker, A., A. Chen, M. Agrez, R.I. Cone, S. Nishimura, E. Wayner, R. Pytela, and D. Sheppard. 1994. Role of the integrin alpha v beta 6 in cell attachment to fibronectin. Heterologous expression of intact and secreted forms of the receptor. J. Biol. Chem. 269:69406948.
Related Article