Hemicentin-1 is An Essential Extracellular Matrix Component of the Dermal-Epidermal and Myotendinous Junctions

Abstract

The extracellular matrix (ECM) architecture is composed of supramolecular fibrillar networks that define tissue specific cellular microenvironments. Hemicentins (Hmcn1 and Hmcn2) are ancient and very large members (>600 kDa) of the fibulin family, whose short members are known to guide proper morphology and functional behavior of specialized cell types predominantly in elastic tissues. However, the tissue distribution and function of Hemicentins within the cellular microenvironment of connective tissues has remained largely unknown. Performing in situ hybridization and immunofluorescence analyses, we found that Hmcn1 and Hmcn2 show a complementary distribution throughout different tissues and developmental stages. In postnatal dermal-epidermal junctions (DEJ) and myotendinous junctions (MTJ), Hmcn1 is primarily produced by mesenchymal cells (fibroblasts, tenocytes), Hmcn2 by cells of epithelial origin (keratinocytes, myocytes). Hmcn1-/-mice are viable and show no overt phenotypes in tissue tensile strength and locomotion tests. However, transmission electron microscopy revealed ultrastructural basement membrane (BM) alterations at the DEJ and MTJ of Hmcn1-/-mice, pointing to a thus far unknown role of Hmcn1 for BM and connective tissue boundary integrity.

Vertebrate extracellular matrix protein hemicentin-1 interacts physically and genetically with basement membrane protein nidogen-2

“…(U-X) Transmission electron micrographs of dermal-epidermal junctions in dorsal median fin fold of wild-type, nid2a morphant, hmcn1 tq102 or lama5 tc17 mutant embryo, 30 hpf, transverse section through tail region; inset show low magnification overviews of dorsal halves of sections, also illustrating the increased width of the dermal space between the two adjacent epidermal sheets of the dorsal median fin fold in nid2a morphant and hmcn1 and lama5 mutants. Arrows point to BM of normal morphology, empty arrow heads in R,T point to regions with indistinct BM in nid2a morphant and lama5 mutant, and filled arrowhead in S points to regions with widened BM in hmcn1 mutant, consistent with the recently reported BM defects in Hmcn1 mutant mice (49).…”

“…To address whether hemicentins and nidogens interact functionally in vivo , we carried out comparative and combined loss-of-function studies. Mouse Hmcn1 mutants only show very mild defects in dermal-epidermal and mytendinous junctions (49), while defects do not seem further enhanced upon combined loss of Hmcn1 and Hmcn2 (48). This largely dispensable nature of mouse hemicentins precludes comparative and combined hemicentin-nidogen loss of function studies, and we therefore turned to zebrafish.…”

“…Thus, only the combined loss of both paralogs leads to blistering defects as strong as those of hmcn1 and lama5 mutants (Supplementary Figure S5), consistent with the partially redundant roles of Nid1 and Nid2 in the mouse (31, 32). Also, at the ultrastructural level, mouse (49) and zebrafish Hmcn1 / hmcn1 mutants display a dermal extension of BM material not apparent upon loss of nid2a or lama5 (Figure 6Q-X), pointing to an additional, possibly Nid2a and Lama5-independent or even Nid2a and Lama5-antagonistic role of Hmcn1 to spatially restrict BM formation. Similarly, for the BM-epidermal junction, hmcn1 mutants develop epidermal integrity defects significantly later (48 hpf) (50) than nid2a morphants and lama5 mutants (30 hpf; Figure 6Z,Z’), pointing to additional functions of laminins and nidogens that are at least partly independent of Hmcn1.…”

“…In invertebrates like the C. elegans (in which hemicentin was were initially discovered, also referred to as him-4 ) (10,12,44) and planarian flatworms (45, 46), they are encoded by a single gene. In vertebrates like mammals (47–49) and zebrafish (50, 51), two paralogs exist, Hmcn1/hmcn1 , also named fibulin-6 , and Hmcn2/hmcn2 . Hemicentins share a number of structural motifs (see Figure 1A): an amino-terminal von Willebrand type A (VWA) domain, followed by a long (>40) stretch of tandem immunoglobulin (Ig) domains, a G2F domain combined with multiple tandem epidermal growth factor (EGF) domains and a fibulin carboxy-terminal module (FC).…”

Inter-donor variability of extracellular matrix production in long-term cultures of human fibroblasts

“…The highest correlations observed for non-collagenous proteins was for THSD4 ( r = 0.95), which already correlated moderately with hydroxyproline content ( r = 0.52), and Hemicentin-1 (HMCN1) ( r = 0.92), a little-studied protein of the fibulin family (Fibulin-6) with roles in epithelial organization. 45–47…”