Fox‐2 protein regulates the alternative splicing of scleroderma‐associated lysyl hydroxylase 2 messenger RNA

The biosynthesis and assembly of the triple helical collagen fibrils is a multi-step process that begins inside a cell but is completed in the extracellular matrix (ECM) (1). One of the important groups of enzymes that play a key role in the collagen biosynthesis pathway is the lysyl hydroxylase (LH) family that includes LH1, LH2 and LH3 (also described as PLOD1, 2 and 3; procollagen lysine oxoglutarate dehydrogenases). These enzymes catalyze the conversion of specific lysine residues (Lys) on the nascent collagen chains to hydroxylysines (Hyl). The target sequences of LH are located in both the helical and non-helical telopeptide collagen domains (Gly-X-Lys and Ala/Ser-X-Lys, respectively) (2). The telopeptide hydroxylysines in turn act as precursors that are responsible for the formation of inter-molecular tri-functional pyridinoline cross-links (Pyr). These Pyr cross-links are critical for the mechanical stability and tensile strength of collagen fibrils. Although each member of the LH family hydroxylates collagen lysine residues, only LH2 has been reported to act specifically on the telopeptide lysines of collagen molecules (3). LH2 is also the only member of the LH family that is known to be alternately spliced (4). Its alternative splicing leads to the generation of two mRNA transcripts, LH2(long) and LH2(short), that either include or exclude the 63bp exon 13A (4, 5). The identification of LH2(long) in dermal fibroblasts and its differential expression in various tissues was first reported by our laboratory (4, 6). Subsequent studies have reported that LH2 (long) mRNA is significantly increased in scleroderma and other fibrotic conditions (7, 8). LH2(long) over-expression is accompanied by an associated increase in the Pyr cross-links present in the accumulated collagen in the scleroderma patients (9–13). Therefore factors affecting the changes in LH2 alternative splicing pattern appear to play an important role in scleroderma. Our current study has established that one of these regulatory factors, Fox-2, promotes the generation of LH2(long) mRNA by enhancing the inclusion of the LH2 alternative exon 13A. We have utilized a new LH2 minigene that recapitulates the endogenous LH2 splicing pattern to identify the relative contributions of the four Fox-binding motifs that are present in the introns flanking the alternately-spliced exon 13A. Using site-directed mutagenesis and Fox-2 over-expression experiments we have demonstrated that the two Fox motifs located in the upstream intron are required for the efficient inclusion of exon 13A. Targeting the expression of Fox-2 protein in primary cultured cells derived from scleroderma biopsy samples using Fox-2 specific siRNAs significantly reduced the endogenous levels of LH2(long) mRNA that are elevated in scleroderma. Our results not only shed new light on the pathobiology of scleroderma, but may indicate a future approach towards development of new strategies that decrease the levels of fibrosis-associated LH2(long) in scleroderma.