Neurite outgrowth by the alternatively spliced region of human tenascin-C is mediated by neuronal alpha7beta1 integrin - PubMed (original) (raw)

Neurite outgrowth by the alternatively spliced region of human tenascin-C is mediated by neuronal alpha7beta1 integrin

Mary Lynn T Mercado et al. J Neurosci. 2004.

Abstract

The region of tenascin-C containing only alternately spliced fibronectin type-III repeat D (fnD) increases neurite outgrowth by itself and also as part of tenascin-C. We previously localized the active site within fnD to an eight amino acid sequence unique to tenascin-C, VFDNFVLK, and showed that the amino acids FD and FV are required for activity. The purpose of this study was to identify the neuronal receptor that interacts with VFDNFVLK and to investigate the hypothesis that FD and FV are important for receptor binding. Function-blocking antibodies against both alpha7 and beta1 integrin subunits were found to abolish VFDNFVLK-mediated process extension from cerebellar granule neurons. VFDNFVLK but not its mutant, VSPNGSLK, induced clustering of neuronal beta1 integrin immunoreactivity. This strongly implicates FD and FV as important structural elements for receptor activation. Moreover, biochemical experiments revealed an association of the alpha7beta1 integrin with tenascin-C peptides containing the VFDNFVLK sequence but not with peptides with alterations in FD and/or FV. These findings are the first to provide evidence that the alpha7beta1 integrin mediates a response to tenascin-C and the first to demonstrate a functional role for the alpha7beta1 integrin receptor in CNS neurons.

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Figures

Figure 1.

Figure 1.

Structure of human tenascin-C. A, Organization of the tenascin-C molecule. This diagram is adapted from Aukhil et al. (1993). The N termini of three arms are joined to form a trimer, and two trimers are connected via a disulfide bond (S-S) to form a hexamer. Each arm consists of 14 domains with homology to epidermal growth factor (EGF), 8-17 FN-III repeats depending on alternative RNA splicing, and a single fibrinogen (fbg) domain. The universal FN-III repeats (fn1-5 and fn6-8) are present in all tenascin-C splice variants. The largest tenascin-C splice variant contains nine alternatively spliced FN-III repeats (designated A1, A2, A3, A4, B, AD1, AD2, C, and D, or fnA-D), which are missing in the shortest splice variant. B, Crystal structure of universal tenascin-C FN-III domain 3 (fn3). This structure is adapted from Leahy et al. (1992) and consists of six exposed loops and seven β strands. The α9β1 integrin recognition sequence EIDGIELT, which corresponds to the neurite outgrowth-promoting sequence VFDNFVLK in fnD, is highlighted and includes portions of an exposed loop and adjacent β strand.

Figure 2.

Figure 2.

A β1 integrin neuronal receptor is implicated in promotion of neurite outgrowth by fnD and VFDNFVLK. A, Rat cerebellar granule neurons were cultured for 24 hr on PLL-coated glass coverslips in the presence of bound fnD or VFDNFVLK and a function-blocking antibody againstβ1 integrin subunit (10 μg/ml). Distributions of total neurite length are presented as a box-and-whisker plot. One representative experiment of four is shown. Boxes enclose 25th and 75th percentiles of each distribution and are bisected by the median; whiskers indicate 5th and 95th percentiles. FnD and VFDNFVLK significantly enhanced outgrowth compared with the PLL control (*p < 0.05; Kolmogorov-Smirnov test). The β1 integrin antibody did not alter neurite outgrowth on PLL but completely blocked outgrowth promotion by fnD and VFDNFVLK. B, Neurons were cultured for 24 hr on PLL-coated coverslips in the presence of bound plus excess soluble (bd + sl) VFDVFVLK and β1 integrin antibody. One representative experiment of three is shown. Outgrowth values were higher than those observed in A for neurons cultured on bound peptide alone and reduced to control values in the presence of β1 antibody.

Figure 3.

Figure 3.

Neurons cultured on bound VFDNFVLK but not VSPNGSLK demonstrate clustering of immunoreactivity for β1 integrin subunit. Immunocytochemistry was performed using a monoclonal hamster antibody against β1 integrin chain, followed by a fluorescein-conjugated goat anti-hamster secondary antibody. VFDNFVLK but not VSPNGSLK induced clustering of neuronal β1 integrin immunoreactivity. Scale bar, 10 μm.

Figure 4.

Figure 4.

Cerebellar granule neurons express the X2B isoform of the α7 integrin subunit. A, Homogenate from P8 rat cerebellar granule neurons (25 μg) was separated on 8% SDS-PAGE gels under nonreducing conditions and then transferred to nitrocellulose paper. Blots were probed with monoclonal O26 antibody, which cross-reacts with all splice variants of the α7 integrin chain, or polyclonal α7A or α7B antibodies, which cross-react with variants containing the A or B cytoplasmic tail. Immunoreactive bands were visualized using the enhanced chemiluminescence method. Blots probed with the O26 or α7B antibody demonstrated one predominant polypeptide band with _M_r of 120,000, whereas blots probed with the α7A antibody revealed no polypeptide band. B, RT-PCR was performed on total RNA prepared from P8 rat cerebellar granule neurons. Amplification was done using sense and antisense primers designed to amplify X1, X2, A, and B variants of the α7 integrin as described in Materials and Methods. The 200 bp fragment corresponds to the X2 extracellular variant, and the 370 bp fragment corresponds to the B cytoplasmic variant. No products were obtained that corresponded to the X1 extracellular variant (220 bp) or the A cytoplasmic variant (480 bp).

Figure 5.

Figure 5.

The α7 integrin subunit is implicated as the partner for β1 in promotion of neurite outgrowth by VFDNFVLK. Rat cerebellar granule neurons were cultured for 24 hr on PLL-coated glass coverslips in the presence of bound VFDNFVLK and function-blocking O26 antibody against α7 integrin subunit (100, 50, 10, 5, 1, or 0.5 μg/ml). One representative experiment of three is shown. VFDNFVLK significantly enhanced outgrowth compared with the PLL control (*p < 0.05; Kolmogorov-Smirnov test). The α7 integrin antibody did not alter neurite outgrowth on PLL at any concentration used. The antibody inhibited promotion of outgrowth by VFDNFVLK and demonstrated a biphasic concentration dependence. Outgrowth was significantly impaired at 50, 10, and 5 μg/ml O26 antibody (**p < 0.05; Kolmogorov-Smirnov test), with maximal inhibition at 10 μg/ml.

Figure 6.

Figure 6.

The α1, α2, α3, and α6 integrin subunits do not play a role in VFDNFVLK-facilitated neurite growth. Rat cerebellar granule neurons were cultured for 24 hr on PLL-coated coverslips in the presence of bound VFDNFVLK and function-blocking antibodies against α1, α2, or α3 integrin subunits (10 μg/ml) (A), whereas mouse cerebellar granule neurons were cultured in the presence of bound VFDNFVLK and a function-blocking antibody against α6 integrin subunit (10 μg/ml) (B). One representative experiment of three is shown. VFDNFVLK significantly enhanced outgrowth compared with PLL (*p < 0.05; Kolmogorov-Smirnov test). None of the antibodies altered neurite outgrowth on PLL or VFDNFVLK.

Figure 7.

Figure 7.

α7 integrin is involved in neurite outgrowth promotion by tenascin-C and laminin-1 but not by fibronectin. A, Rat cerebellar granule neurons were cultured for 24 hr on PLL-coated coverslips in the presence of bound large tenascin-C and function-blocking antibody O26 against α7 integrin subunit or function blocking antibody against β1 integrin subunit. One representative experiment of three is shown. Tenascin-C significantly enhanced outgrowth compared with PLL (*p < 0.05; Kolmogorov-Smirnov test). The α7 antibody partially blocked outgrowth promotion by large tenascin-C; the reduction in outgrowth values was significant (**p < 0.05; Kolmogorov-Smirnov test). The β1 integrin antibody completely blocked outgrowth promotion by large tenascin-C. B, Rat cerebellar granule neurons were cultured for 24 hr on PLL-coated coverslips in the presence of bound laminin-1 or fibronectin and function-blocking O26 antibody against α7 integrin subunit. One representative experiment of three is shown. Laminin-1 and fibronectin significantly enhanced outgrowth compared with PLL (*p < 0.05; Kolmogorov-Smirnov test). The α7 antibody partially reduced neurite outgrowth on laminin-1; the reduction was significant (**p < 0.05; Kolmogorov-Smirnov test). The antibody did not alter neurite outgrowth on fibronectin.

Figure 8.

Figure 8.

Neuronal α7β1 integrin binds to D5 peptide in an FD-FV-dependent manner. P8 cerebellar granule neuronal extracts were incubated with Sepharose beads conjugated with either wild-type D5 peptide (D5 wt) or D5 mutant 1 peptide with alterations in both FD and FV (D5 mt). A, Proteins eluted with EDTA from wild-type or mutant D5 peptide were separated on 8% SDS-PAGE gels under nonreducing conditions and transferred to nitrocellulose paper. Blots were probed with monoclonal O26 antibody against α7 integrin chain or polyclonal antibody against β1 integrin chain. Immunoreactive bands were visualized using the enhanced chemiluminescence method. Blots of proteins eluted with EDTA from D5 wt peptide revealed polypeptide bands with _M_r of 120,000 and 116,000, respectively, for the O26 and β1 integrin antibodies. Blots of proteins eluted with EDTA from D5 mt peptide revealed no polypeptide bands. B, Silver-stained gels revealed overlapping polypeptide bands with _M_r of 120,000 and 116,000 for proteins eluted with EDTA from D5 wt peptide but not D5 mt 1 peptide.

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