Expression of Sulf1 and Sulf2 in cartilage, bone and endochondral fracture healing

Abstract

Highlights: High Sulf1/Sulf2 expression in active osteoblasts with reduction in osteocytes. High variability in Sulf1/Sulf2 expression in articular chondrocytes. Hypertrophic chondrocytes in growing and healing bone express high levels of Sulf2 but not Sulf1. High Sulf2 expression in growing and healing bone closely correlates with Hedgehog signalling.ABSTRACT: SULF1/SULF2 enzymes regulate cell signalling that impacts the growth and differentiation of many tissues. To determine their possible role in cartilage and bone growth or repair, their expression was examined during development and bone fracture healing using RT-PCR and immunochemical analyses. Examination of epiphyseal growth plates revealed differential, inverse patterns of SULF1 and SULF2 expression, with the former enriched in quiescent and the latter in hypertrophic chondrocyte zones. Markedly higher levels of both SULFs, however, were expressed in osteoblasts actively forming bone when compared with proliferating pre-osteoblasts in the periosteum or the entombed osteocytes which express the lowest levels. The increased Sulf1 and Sulf2 expression in differentiating osteoblasts was further confirmed by RT PCR analysis of mRNA levels in rat calvarial osteoblast cultures.SULF1 and SULF2 were expressed in most fetal articular chondrocytes but downregulated in a larger subset of cells in the postnatal articular cartilage. Unlike adult articular chondrocytes, SULF1/SULF2 expression varied markedly in postnatal hypertrophic chondrocytes in the growth plate, with very high SULF2 expression compared with SULF1 apparent during neonatal growth in both primary and secondary centres of ossification. Similarly, hypertrophic chondrocytes expressed greatly higher levels of SULF2 but not SULF1 during bone fracture healing. SULF2 expression unlike SULF1 also spread to the calcifying matrix around the hypertrophic chondrocytes indicating its possible ligand inhibiting role through HSPG desulfation. Higher levels of SULF2 in both developing and healing bone closely correlated with parallel increases in hedgehog signalling analysed by ptc1 receptor expression.

SULF1/SULF2 reactivation during liver damage and tumour growth

“…This is compatible with increased expression of Sulfs during fetal development of many tissues (Gill et al 2010;Hitchins et al 2013) and during the development of different tumour types (Gill et al 2012;Gill et al 2014;Lemjabbar-Alaoui et al). It, however, differs from some other studies reporting Sulf1 to be a tumour suppressor in liver (Lai et al 2006;Lai et al 2008a;Xu G et al 2014) thus implying higher level of expression in normal adult liver although some other adult tissues such as neuronal and skeletal tissues have been reported to continue expressing Sulf1/Sulf2 throughout life (Joy et al 2015;Zaman et al 2016). Sulf1 activation, however is not unique to fetal or tumour growth since a number of other conditions including injury-induced repair can also activate these enzymes.…”

Comparative and Evolutionary Studies of Vertebrate Extracellular Sulfatase Genes and Proteins: SULF1 and SULF2

“…SULF1 and SULF2 are secreted enzymes which carry out essential roles in the extracellular environment by catalysing endoglucosamine-6-sulfatase activity and removing 6-O sulfate groups from Heparin Sulfate Proteoglycans (HSPGs) [3,4]. These perform several key roles: modulating the activity of growth factor receptors and cell signaling pathways, such as FGF, VEGF, GDNF and WNT signaling pathways, which initiate gene transcription signals through cell surface receptors [5][6][7][8][9][10][11]; serving essential roles in vertebrate development [12][13][14][15][16][17][18][19][20]; and in modulating microbial (Chlamydia muridarum) infection [21].…”

“…Human SULF1, which spans 194.3 kilobases and comprises 22 exons, is localized on chromosome 8; whereas human SULF2 spans 128.7 kilobases and comprises 21 exons on chromosome 20 [26,27]. Both of these genes are widely expressed in the body, consistent with their overlapping and essential roles in cell signaling pathways, skeletal muscle regeneration, neonatal development and survival, metastasis and wound repair [9,18,19,23]. This paper reports the predicted gene structures and amino acid sequences for several vertebrate SULF1 and SULF2 genes and proteins, the predicted secondary and tertiary structures for human SULF1 and SULF2 protein subunits, and the structural, phylogenetic and evolutionary relationships for these genes and enzymes.…”

Short SULF1/SULF2 splice variants predominate in mammary tumours with a potential to facilitate receptor tyrosine kinase-mediated cell signalling

“…Equivalent amount of total RNA (1 μg) for each sample was reverse-transcribed into cDNA using SuperScript II reverse transcriptase (Invitrogen), using random primers (Invitrogen, Paisley, UK) for RT-PCR analysis. The PCR of cDNA was carried out using primers to exons 5 and 9 of the catalytic domain that we have shown to splice out exons 6-8 in different combinations in our earlier study (Gill et al 2014;Zaman et al 2016). The primers: 5'-CGAGGTTCAGAGGACGGATA-3' and 5'-GCCTCTCCACAGAATCATCC-3' were used to amplify 804-bp fragment of catalytic domain, nucleotides 83-886 bp of Sulf1.…”

“…The primers: 5'-CGAGGTTCAGAGGACGGATA-3' and 5'-GCCTCTCCACAGAATCATCC-3' were used to amplify 804-bp fragment of catalytic domain, nucleotides 83-886 bp of Sulf1. Primers, 5'-CAACTGTGTTCTCCCTGCTGGGT-3' & 5'-CTGGAGCATGTTGGTGAATTCC-3′ were used to amplify a region of Sulf2 catalytic domain, nucleotides 38-843 (Zaman et al 2016). PCR fragments following 40 amplification cycles separated in 2% agarose gels were cut out and purified to verify their identity by DNA sequencing (GATC Biotech).…”