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T J Hornyak

Publications and source records attributed to T J Hornyak.

8 recordsLinked to original sources

Analysis of SOX10 function in neural crest-derived melanocyte development: SOX10-dependent transcriptional control of dopachrome tautomerase.

SOX10 is a high-mobility-group transcription factor that plays a critical role in the development of neural crest-derived melanocytes. At E11.5, mouse embryos homozygous for the Sox10(Dom) mutation entirely lack neural crest-derived cells expressing the lineage marker KIT, MITF, or DCT. Moreover, neural crest cell cultures derived from homozygous embryos do not give rise to pigmented cells. In contrast, in Sox10(Dom) heterozygous embryos, melanoblasts expressing KIT and MITF do occur, albeit in reduced numbers, and pigmented cells eventually develop in nearly normal numbers both in culture and in vivo. Intriguingly, however, Sox10(Dom)/+ melanoblasts transiently lack Dct expression both in culture and in vivo, suggesting that during a critical developmental period SOX10 may serve as a transcriptional activator of Dct. Indeed, we found that SOX10 and DCT colocalized in early melanoblasts and that SOX10 is capable of transactivating the Dct promoter in vitro. Our data suggest that during early melanoblast development SOX10 acts as a critical transactivator of Dct, that MITF, on its own, is insufficient to stimulate Dct expression, and that delayed onset of Dct expression is not deleterious to the melanocyte lineage.

Animals↗

Transcription factors in melanocyte development: distinct roles for Pax-3 and Mitf.

A transgenic mouse model was used to examine the roles of the murine transcription factors Pax-3 and Mitf in melanocyte development. Transgenic mice expressing beta-galactosidase from the dopachrome tautomerase (Dct) promoter were generated and found to express the transgene in developing melanoblasts as early as embryonic day (E) 9.5. These mice express the transgene in a pattern characteristic of endogenous Dct expression. Transgenic mice were intercrossed with two murine coat color mutants, Splotch (Sp), containing a mutation in the murine Pax3 gene, and Mitf(mi), with a mutation in the basic-helix-loop-helix-leucine zipper gene Mitf. Transgenic heterozygous mutant animals were crossed to generate transgenic embryos for analysis. Examination of beta-galactosidase-expressing melanoblasts in mutant embryos reveals that Mitf is required in vivo for survival of melanoblasts up to the migration staging area in neural crest development. Examination of Mitf(mi)/+ embryos shows that there are diminished numbers of melanoblasts in the heterozygous state early in melanocyte development, consistent with a gene dosage-dependent effect upon cell survival. However, quantification and analysis of melanoblast growth during the migratory phase suggests that melanoblasts then increase in number more rapidly in the heterozygous embryo. In contrast to Mitf(mi)/Mitf(mi) embryos, Sp/Sp embryos exhibit melanoblasts that have migrated to characteristic locations along the melanoblast migratory pathway, but are greatly reduced in number compared to control littermates. Together, these results support a model for melanocyte development whereby Pax3 is required to expand a pool of committed melanoblasts or restricted progenitor cells early in development, whereas Mitf facilitates survival of the melanoblast in a gene dosage-dependent manner within and immediately after emigration from the dorsal neural tube, and may also directly or indirectly affect the rate at which melanoblast number increases during dorsolateral pathway migration.

Animals↗

Cell-density-dependent regulation of expression and glycosylation of dopachrome tautomerase/tyrosinase-related protein-2.

The expression of the dopachrome tautomerase gene (Dct) and its protein product, tyrosinase-related protein-2, was studied in the cultured, phorbol-ester-dependent murine melanocyte cell line melan-a. Increased cell density was found to stimulate Dct expression both in cells stably transfected with a Dct promoter-lacZ construct and endogenously in nontransfected cells. Increased Dct expression under these conditions corresponds to increased tyrosinase-related protein-2 production. Tyrosinase-related protein-2 was found to exist in two distinct glycoforms with different endoglycosidase sensitivities. Density-dependent expression of tyrosinase-related protein-2 was independent of time of cell growth, cell proliferation, and soluble factors, implying that cell-cell contact is the important determinant governing increased Dct expression under these conditions. Tyrp1 gene expression and tyrosinase-related protein-1 production were also induced under similar conditions. The results show that cell-cell contact between melanocytes induces a coordinated response at both transcriptional and nontranscriptional levels that induces production of the tyrosinase-related proteins that have a significant role in melanization.

Animals↗

Interactions of factor XIII with fibrin as substrate and cofactor.

Factor XIIIa (a2') is a homodimeric transglutaminase that is formed via limited alpha-thrombin-catalyzed proteolysis of the platelet (a2) or plasma (a2b2) factor XIII zymogen in a reaction that results in proteolytic removal of a 37-aminoacyl residue peptide from the N-terminus of the a chains and exposure of the active-site thiol group in the resulting a' chains of factor XIIIa. In this study, we characterized interactions of factor XIII and factor XIIIa with fibrin, a natural substrate for factor XIIIa and a cofactor for the alpha-thrombin-catalyzed activation of plasma factor XIII. The carbamylmethyl derivatives of the active-site thiol group of platelet factor XIII (CMa2) and factor XIIIa (CMa2') were prepared, and their interactions with fibrin were measured. The enzyme-like derivative (CMa2') which contained nicked a' chains bound more tightly to fibrin (Kd = 2.1 microM) than did CMa2 (Kd = 14 microM), the platelet zymogen-like derivative with intact a chains, but the binding of each was weaker than the binding of plasma factor XIII zymogen (a2b2) to fibrin (Kd = 0.20 microM) under the same conditions. Saturation of fibrin with plasma factor XIII zymogen (a2b2) did not affect the binding of CMa2' to fibrin, suggesting that the plasma factor XIII zymogen (a2b2) and the active-site-modified form of factor XIIIa (CMa2') bind to separate, noninteracting sites of fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗

Role of calcium ion in the generation of factor XIII activity.

The involvement of calcium ion in the activation of both plasma factor XIII (alpha 2 beta 2) and platelet factor XIII (alpha 2) was investigated. The second-order dependence of the rate constant for exposure of the active-site thiol group of alpha-thrombin-cleaved plasma factor XIII (alpha 2'beta 2) on the concentration of calcium ion suggested that the binding of two calcium ions is required for transformation of the alpha 2'beta 2 tetramer to enzymatically active factor XIIIa. Fibrinogen, previously reported to lower the calcium ion concentration required for efficient activation of alpha 2'beta 2 [Credo, R. B., Curtis, C. G., & Lorand, L. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4234-4237], was found in the present study to increase the rate of exposure of the active-site thiol group. Whereas calcium ion is required for exposure of the active-site thiol group in cleaved plasma factor XIII (alpha 2'beta 2), exposure of an active-site thiol group in cleaved platelet factor XIII (alpha 2') occurs in the absence of calcium ion. The rate constant (2.2 x 10(5) M-1 s-1) for alpha-thrombin-catalyed exposure of the active-site thiol group of platelet factor XIII zymogen (alpha 2) in the presence of calcium ion was greater than the rate constant (0.7 x 10(5) M-1 s-1) determined in the absence of calcium ion.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗

Alpha-thrombin-catalyzed activation of human platelet factor XIII: relationship between proteolysis and factor XIIIa activity.

The kinetics of activation of platelet factor XIII, an a-subunit dimer, were characterized by determining rate constants for activation peptide (AP) release, generation of activity, and exposure of the active-site thiol group. The specificity constant (kappacat/Km) for alpha-thrombin-catalyzed AP release, 1.2 x 10(5) M-1s-1, was found to be similar to that for AP release from the tetramer plasma factor XIII (a2b2) [Janus, T.J., Lewis, S. D., Lorand, L., & Shafer, J. A. (1983) Biochemistry 22, 6269-6272], implying that the b subunits of plasma factor XIII do not hinder alpha-thrombin-catalyzed cleavage of AP from the a subunit. Platelet factor XIIIa activity was generated at a rate approximately twice the rate of AP release. This difference in rates was shown to be consistent with a reaction pathway for activation of platelet factor XIII wherein full factor XIIIa activity is generated when one AP is removed from the dimeric zymogen so that removal of the second AP has no detectable effect on catalytic activity. In accord with this conclusion, the rate constant for exposure of the active-site thiol group, as measured by the incorporation of [1-14C]-iodoacetamide, was about twice that observed for the removal of AP.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗