Identification of PINCH in Schwann cells and DRG neurons: Shuttling and signaling after nerve injury

Abstract

Particularly interesting new cysteine-histidine rich protein (PINCH) is a double zinc finger domain (LIM)-only adapter protein that functions to recruit the integrin-linked kinase (ILK) to sites of integrin activation. Genetic studies have shown that PINCH and ILK are required for integrin signaling. Since integrin activation is associated with Schwann cell migration, neurite outgrowth and regeneration, this study examined PINCH in the normal peripheral nervous system and after chronic constriction injury (CCI) in adult Sprague-Dawley rats. Immunohistochemistry identified PINCH immunoreactivity in cell bodies of dorsal root ganglia (DRG) neurons, axons, satellite cells, and Schwann cells. PINCH immunostaining was localized to the membrane of uninjured DRG cell bodies consistent with its localization at a site of integrin activation. In contrast, 5 days following CCI, PINCH immunostaining was diffuse throughout the DRG cell cytoplasm. Confocal microscopy of primary and transformed Schwann cells localized PINCH in cytoplasmic, perinuclear and nuclear areas. Examination of the PINCH sequence revealed a putative leucine-rich nuclear export signal (NES) and an overlapping basic nuclear localization signal (NLS). To demonstrate nuclear export of PINCH, rabbit anti-PINCH IgG was microinjected into Schwann cell nuclei and allowed to combine with PINCH contained within the nucleus. Immunofluorescence showed that the PINCH and anti-PINCH IgG complex rapidly translocated to the cytoplasm. Treatment with leptomycin B caused nuclear accumulation of PINCH, indicating that the CRM1 pathway mediates nuclear export of PINCH. ILK activity in Schwann cells was enhanced by platelet-derived growth factor (PDGF) and tumor necrosis factor alpha. PINCH immunoprecipitates from PDGF- and TNFalpha-stimulated Schwann cells contained several high-molecular-weight threonine-phosphorylated proteins. Taken together, these results indicate that PINCH is an abundant shuttling/signaling protein in Schwann cells and DRG neurons.

PINCH1 regulates cell-matrix and cell-cell adhesions, cell polarity and cell survival during the peri-implantation stage

“…Consistent with such binding properties, the first LIM domain of PINCH has been shown to bind ILK and the fourth LIM domain the SH2/SH3 adaptor protein Nck2 (or Grb4), which in turn can associate with several receptor tyrosine kinases and thereby links integrins to growth factor signalling (Tu et al, 1998). Furthermore, mammalian PINCH1 and 2 and C. elegans PINCH have been shown to be present in the nucleus of a variety of cell types (Hobert et al, 1999;Zhang et al, 2002a;Campana et al, 2003). Whether nuclear PINCH1 and 2 binds nuclear proteins and/or DNA, however, is not known.…”

PINCH Expression and Its Clinicopathological Significance in Gastric Adenocarcinoma

“…The preparation, specificity and reliability of the rabbit polyclonal PINCH antibody used in the study were described previously [7,14]. Five-um sections from paraffin-embedded tissue blocks were deparaffinized, hydrated and rinsed in distilled H 2 O.…”

Migfilin and its binding partners: from cell biology to human diseases

“…Since the migfilin mutant lacking an NES has increased transcription-and differentiation-promoting activities (Akazawa et al, 2004), migfilin(s) probably also has higher transcription-and differentiationpromoting activity. It has been shown that several other LIM-containing focal adhesion proteins, including zyxin (Nix and Beckerle, 1997), LPP (for 'lipoma preferred partner') (Petit et al, 2000), Ajuba (Kanungo et al, 2000), Trip6 (for 'thyroid hormone interacting protein 6') (Wang and Gilmore, 2001), Hic-5 (for 'hydrogen peroxide-inducible clone 5') (Shibanuma et al, 2003;Shibanuma et al, 1997;Yang et al, 2000), PINCH-1 (Campana et al, 2003) and PINCH-2 (Zhang et al, 2002), can also be transported to the nucleus. Although much remains to be learned about their functions in the nucleus, currently available data suggest that they probably recognize different sets of nuclear targets and therefore mediate diverse nuclear activities.…”